Anti-c-met tandem fc bispecific antibodies

ABSTRACT

Provided herein are tandem Fcs and tandem Fc antibodies (“TFcAs”), e.g., tandem Fc bispecific antibodies (“TFcBAs”), which comprise one or at least two binding sites that specifically bind to one or more cell surface receptors. The binding sites are connected through a TFc, which TFc comprises a first Fc region and a second Fc region, wherein the first and the second Fc regions are linked through a TFc linker to form a contiguous polypeptide and dimerize to form an Fc dimer. Exemplary TFcBAs bind to the cell surface receptors c-Met and EpCam and inhibit signal transduction through the cell surface receptor(s) for which the binding sites of the TFcBA are specific.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/199,760, filed Mar. 6, 2014, which claims the benefit of U.S.Provisional Application No. 61/773,764 filed Mar. 6, 2013, and U.S.Provisional Application No. 61/773,788 filed Mar. 6, 2013; all of theforegoing applications are incorporated by reference in entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Sep. 30, 2016, is named MMJ_083CN_Sequence_Listing.txt and is 1,401,428 bytes in size.

BACKGROUND

It has been established that tumor cells express receptors for growthfactors and cytokines that stimulate proliferation of the cells and,moreover, that antibodies to such receptors (e.g., tyrosine kinasereceptors) can be effective in blocking the stimulation of cellproliferation mediated by growth factors and cytokines to inhibit tumorcell growth. Commercially available therapeutic antibodies that targetreceptors on cancer cells include, for example, trastuzumab (Herceptin)for the treatment of breast cancer, which targets the HER2 receptor(also known as ErbB2), and cetuximab (Erbitux) for the treatment ofcolorectal cancer and head and neck cancer, which targets the epidermalgrowth factor receptor (EGFR, also known as HER1 or ErbB1).

While this approach of administering a therapeutic agent comprising onlya single therapeutic monoclonal antibody (when administered in theabsence of administration of another therapeutic antibody, referred toherein as monotherapy) has shown considerable success in cancertreatment, there are a number of factors that can lead to failure ofsuch treatment or recurrence of tumor growth after initial inhibition.For example, certain tumors rely on more than one growth factor-mediatedsignal transduction pathway for cell proliferation and thus targeting ofa single pathway may prove insufficient to significantly affect tumorcell growth. Alternatively, even in cases where one pathway is the onlyor predominant growth-stimulatory pathway, certain tumors cells arecapable of activating another signaling pathway for growth stimulationwhen the original one is blocked by antibody (innate resistance totreatment). Still further, some tumors exhibit initial responsiveness toantibody monotherapy but later develop resistance to treatment byswitching to use of another signaling pathway (acquired resistance totreatment). Furthermore, some receptors, such as a c-Met receptor, arestimulated by conventional antibodies (e.g., IgG antibodies) totransduce signals into a cell upon which they are expressed. Withoutintending to be bound by any particular theory of operation, thisphenomenon is believed to result from the crosslink pairing of tworeceptors on the same cell by the paired, generally identical, antigenbinding sites present on antibodies such as IgAs, IgDs, IgEs, IgGs andIgMs. Such stimulation may have the opposite effect to that whichproduces a therapeutic benefit—e.g., cell growth and replication may beupregulated by such anti-c-Met antibodies, rather than inhibited.

In embodiments, the interaction with a receptor or plurality ofreceptors (e.g., a c-Met receptor) and a composition of the inventionresults in an antagonist activity. In a particular embodiment, suchinteraction results in an antagonist activity and is substantially freeor free of agonist activity. In an aspect, antagonist activity is usefulfor compositions and methods of inhibiting cancer.

Accordingly, additional therapeutic approaches for cancer treatment areneeded to overcome limitations of antibody monotherapy and to provideother benefits.

SUMMARY

Provided herein are engineered antibodies, such as Tandem Fc Antibodies(“TFcAs”). Exemplary TFcAs are Tandem Fc Bispecific Antibodies (TFcBAs)such as those described in U.S. Provisional Application No. 61/527,802,filed Aug. 26, 2011; and copending PCT International Application No.PCT/US2012/052490, filed Aug. 27, 2012 (see Int'l Pub. No.WO2013/033008A2). A TFcBA comprises a Tandem Fc, which is a polypeptidemoiety that comprises a first Fc region and a second Fc region, each ofsaid first Fc region and second Fc region having a C-terminus and anN-terminus; the first Fc region and the second Fc region are linked as asingle polypeptide chain through a TFc linker having a C-terminus and anN-terminus (i.e., the C-terminus of the first Fc region is linked by apeptide bond to the N-terminus of the TFc linker, the C-terminus ofwhich TFc linker is in turn linked by a peptide bond to the N-terminusof the second Fc region). A TFcBA may comprise at least two bindingsites (at least a first binding site and a second binding site). Eachsuch binding site binds specifically to a specific part of a cellsurface receptor. Exemplary cell surface receptors are those that areexpressed or overexpressed by cancer cells. Exemplary binding sitesinclude antibody-derived binding sites that bind immunospecifically toan extracellular domain of a cell surface receptor. The first or thesecond binding site of a TFcA or TFcBA may bind specifically to a humanreceptor protein selected from the group consisting of ErbB2, ErbB3(e.g., a binding site described in U.S. Pat. No. 7,846,440), ErbB4,IGF1R, IGF2R, Insulin receptor, RON, c-Met, EGFR, VEGFR1, VEGFR2, TNFR,FGFR1-4, PDGFR (alpha and beta), c-Kit, EPCAM and EphA2. Generally, thereceptor will be a receptor tyrosine kinase. Typically such binding willbe specific to the extracellular portion of the receptor protein. Incertain embodiments disclosed herein, one of the at least two bindingsites comprised by a TFcBA is a binding site specific to c-Met, e.g., ananti-c-Met Fab or an anti-cMet scFv. In certain exemplified embodimentsa TFcBA is provided that comprises a single anti-c-Met binding site andat least one second binding site that does not bind to c-Met, e.g., abinding site specific to ErbB2, ErbB3, ErbB4, IGF1R, IGF2R, Insulinreceptor, RON, EGFR, VEGFR1, VEGFR2, TNFR, FGFR1-4, PDGFR (alpha andbeta), c-Kit, EPCAM and EphA2, wherein the anti-c-Met binding site andthe second binding site are linked through a TFc to form a contiguouspolypeptide. TFcBAs are provided that bind to two epitopes (e.g.,extracellular epitopes) on a single receptor or to two distinct cellsurface receptors and which, upon such binding, strongly inhibit signaltransduction that is normally stimulated by a cognate ligand of at leastone cell surface receptor to which the TFcBA binds. For example, ananti-c-Met+anti-EGFR TFcBA may inhibit signal transduction induced byeither or both of HGF (hepatocyte growth factor, the cognate ligand ofC-met) and EGF (epidermal growth factor, the cognate ligand of EGFR), oran anti-c-Kit+anti-RON TFcBA may inhibit signal transduction induced byeither or both of Macrophage Stimulating Protein (the cognate ligand ofRON) and Stem Cell Factor (the cognate ligand of c-Kit), or ananti-c-Met+anti-EPCAM TFcBA may inhibit signal transduction induced byHGF and may inhibit signaling by the c-Met receptor in the absence ofHGF (e.g., ligand-independent c-Met signaling); each such inhibitionbeing with an IC₅₀ of 10 nM or less or 1 nM or less or 100 pM or less,or with a maximal percent inhibition of at least 70% or at least 80% orat least 90%, as indicated by inhibition of ligand-induced (or in thecase of c-Met signaling in the absence of HGF, ligand-independent)phosphorylation of the receptor(s) that are signal transductioninhibited by the TFcBA. In many embodiments, anti-c-Met comprisingTFcBAs provided herein produce essentially no stimulatory effect oncells expressing c-Met. In other embodiments, anti-c-Met comprisingTFcBAs provided herein induce downregulation or degradation of c-Metreceptors on cells expressing c-Met. In other embodiments, anti-EPCAMTFcBAs herein provided produce most (e.g., 60%, 70%, 80%, 90% or greaterthan 95%) of their signaling inhibition effects in cells expressing lowEPCAM levels (as few as 100,000+/−5% EPCAM molecules per cell) or verylow EPCAM levels (as few as 24,000+/−5% EPCAM molecules per cell). Incertain embodiments, expression of the TFcBA in a cell produces (i) more(i.e. a greater percentage of) correctly formed TFcAB molecules relativeto the expression of a multivalent antibody that binds to the samereceptor(s) but does not comprise a TFc or (ii) more than 80% ofcorrectly formed TFcAB molecules as determined, e.g., by Size ExclusionChromatography (SEC).

Also provided herein are Abs which are TFcBAs, wherein the TFcBAscomprise a first binding site and a second binding site, wherein thefirst binding site binds to a first target and the second binding sitebinds to a second target, and wherein (i) the first and the secondbinding sites are linked through a TFc; (ii) the TFc comprises a firstFc region and a second Fc region, each said first Fc region and secondFc region having a C-terminus and an N-terminus; (iii) the first Fcregion and the second Fc region are linked through a TFc linker having aC-terminus and an N-terminus to form a contiguous polypeptide; (iv) thefirst and the second Fc regions associate (bind) to form an Fc dimer;and (v) either or both of the first and the second Fc region compriseone or more amino acid (aa) modification to enhance or stabilize thebinding between the first and the second Fc region. The TFcBA mayinhibit signal transduction through either or both of the first and thesecond target. In certain embodiments, expression of the TFcBA in a hostcell produces (i) more correctly formed TFcAB molecules relative to theexpression in a matched host cell of a multivalent antibody that bindsto the same receptor(s) but does not comprise a TFc or (ii) more than80% of correctly formed TFcBA molecules as determined, e.g., by SEC.

Further provided herein are monovalent tandem FC antibodies (TFcAs). Amonovalent TFcA may comprise a single binding site that binds to atarget, wherein the binding site is linked to a TFc comprising a firstFc region and a second Fc region, each said first Fc region and secondFc region having a C-terminus and an N-terminus; and wherein (i) thefirst Fc region and the second Fc region are linked through a TFc linkerhaving a C-terminus and an N-terminus to form a contiguous polypeptide;(ii) the first and the second Fc regions associate to form an Fc dimer;and (iii) either or both of the first and the second Fc region compriseone or more aa modification to enhance or stabilize the binding betweenthe first and the second Fc region. The monovalent TFcA may inhibitsignal transduction through the target. In certain embodiments,expression of the monovalent TFcA in a host cell produces (i) morecorrectly formed TFcA molecules relative to the expression in a matchedhost cell of an antibody that does not comprise a TFc or (ii) more than80% of correctly formed TFcA molecules as determined, e.g., by SEC.

The first Fc region and the second Fc region of a TFc comprised by aTFcA, such as a TFcBA, may comprise a first and a second CH3 domain,respectively, each said CH3 domain having a C-terminus and anN-terminus. The first and the second Fc regions of a TFc comprised by aTFcA may comprise a first and a second CH2 domain, respectively, eachsaid CH2 domain having a C-terminus and an N-terminus. The first and thesecond Fc regions of a TFc comprised by a TFcA may comprise a first anda second hinge, respectively, each said first hinge and said secondhinge having a C-terminus and an N-terminus. In certain embodiments, thesecond hinge does not comprise an upper hinge subdomain. The TFccomprised in the TFcA may comprise in amino to carboxyl terminal order:a first CH2 domain, a first CH3 domain, a TFc linker, a second CH2domain and a second CH3 domain. The TFc comprised in the TFcA maycomprise in amino to carboxyl terminal order: a first hinge, a first CH2domain, a first CH3 domain, a TFc linker, a second CH2 domain and asecond CH3 domain. The TFc comprised in the TFcA may comprise in aminoto carboxyl terminal order: a first hinge, a first CH2 domain, a firstCH3 domain, a TFc linker, a second hinge, a second CH2 domain and asecond CH3 domain. The first hinge may comprise an upper hingesubdomain, a core hinge subdomain and a lower hinge subdomain and thesecond hinge may comprise a core hinge subdomain and a lower hingesubdomain, but not an upper hinge subdomain, each said hinge sub-domainhaving a C-terminus and an N-terminus. The TFc comprised by the TFcA maycomprise in amino to carboxyl terminal order: a first hinge, which islinked at its C-terminus to the N-terminus of a first CH2 domain, whichis linked at its C-terminus to the N-terminus of a first CH3 domain,which is linked at its C-terminus to the N-terminus of a TFc linker,which is linked at its C-terminus to the N-terminus of a second hinge,which is linked at its C-terminus to the N-terminus of a second CH2domain, which is linked at its C-terminus to the N-terminus of a secondCH3 domain.

A TFc linker of a TFc comprised by a TFcA may comprise 20-50 aas. A TFclinker may be a Gly-Ser linker, such as (Gly₄Ser)_(n), wherein n is 4,5, 6, 7 or 8. A TFc linker may also comprise an aa sequence that is atleast about 70%, 80%, 90%, 95%, 97%, 98%, or 99% identical to an aasequence of a Gly-Ser linker or which differs therefrom in at most 20,15, 10, 5, 4, 3, 2, or 1 aa addition, deletion or substitution.

A TFc of a TFcA may be an IgG1 TFc. A TFc may be a hybrid TFc, e.g., anIgG1/IgG4 hybrid TFc. A TFc of a TFcA may be an IgG1 TFc and maycomprise in amino to carboxyl terminal order: a first IgG1 hinge, afirst IgG1 CH2 domain, a first IgG1 CH3 domain, a TFc linker, a secondIgG1 hinge, a second IgG1 CH2 domain, and a second IgG1 CH3 domain. Ahybrid TFc may comprise in amino to carboxyl terminal order: a firstIgG1/IgG4 hinge, a first IgG4 CH2 domain, a first IgG1 CH3 domain, a TFclinker, a second IgG4 hinge, a second IgG4 CH2 domain, and a second IgG1CH3 domain.

Either or both of the first CH3 domain and the second CH3 domain of aTFc may comprise one or more aa modifications that enhance or stabilizethe binding between the first and the second Fc regions, as evidenced,e.g., by an essentially uniform product (or band) on a non-denaturingSDS-Page gel. Each of the first CH3 domain and the second CH3 domain ofa TFc may comprise an amino acid modification, which modification is anAssociation Enhancing Modification (“AEM”) that enhances the associationof the first CH3 domain with the second CH3 domain. An AEM may becomprised by a module selected from the group consisting of AEM module1, AEM module 2, AEM module 3 and AEM module 4. Either or both of thefirst Fc region and the second Fc region of a TFc may comprise an aamodification that adds a cysteine as an insertion or replacement, whichcysteine forms a disulfide bond with a cysteine in the other Fc region(a “DiS” modification). Either or both of the first and the second Fcregion of a TFc may comprise a DiS modification in a hinge. In certainembodiments, either or both of the first and the second Fc regioncomprise a DiS modification in a CH3 domain. The DiS modification may becomprised by DiS module 1 or DiS module 2. Each of the first CH3 domainand the second CH3 domain of a TFc may comprise one or more AEMmodifications and one or more DiS modifications.

Either or both of the first and the second CH3 domains of a TFc maycomprise an aa sequence that is at least 70%, 80%, 90%, 95%, 97%, 98%,or 99% identical to an aa sequence of a CH3 domain provided herein,e.g., selected from the group consisting of SEQ ID NOs:27-98, or whichdiffers therefrom in at most 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 aaadditions, deletions or substitutions. In certain embodiments, if the aasequence of a CH3 domain is not identical to a sequence selected fromthe group of sequences SEQ ID NOs:27-98, then the aa sequence of the CH3domain nevertheless comprises the particular AEM and/or DiS of thesequence to which it is similar. The first CH3 domain or the second CH3domain of a TFc may comprises an aa sequence provided herein, e.g.,selected from the group consisting of SEQ ID NOs:27-98. The first CH3and second CH3 domains of a TFc together may comprise a pair of twodifferent members, each member being a CH3 aa sequence, each pairselected from the group of pairs consisting of SEQ ID NOs:31 and 35; SEQID NOs:33 and 37; SEQ ID NOs:39 and 43; SEQ ID NOs:41 and 45; SEQ IDNOs:47 and 51; SEQ ID NOs:49 and 53; SEQ ID NOs:55 and 59; SEQ ID NOs:57and 61; SEQ ID NOs:63 and 67; SEQ ID NOs:65 and 69; SEQ ID NOs:71 and73; SEQ ID NOs:72 and 74; SEQ ID NOs:75 and 79; SEQ ID NOs:77 and 81;SEQ ID NOs:83 and 85; SEQ ID NOs:84 and 86; SEQ ID NOs:87 and 89; SEQ IDNOs:88 and 90; SEQ ID NOs:91 and 93; SEQ ID NOs:92 and 94; SEQ ID NOs:95and 97; and SEQ ID NOs:96 and 98, each member aa sequence being at least70%, 80%, 90%, 95%, 97%, 98%, or 99% identical to, or differing in atmost 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 aa additions, deletions orsubstitutions from the each sequence of each said pair, wherein thefirst CH3 domain comprises a different member of the pair than iscomprised by the second CH3 domain. The first and the second CH3 domainsof a TFc may each comprise an aa sequence that identical to an aasequence of a member of the pair of CH3 aa sequences selected from thegroup consisting of SEQ ID NOs:31 and 35; SEQ ID NOs:33 and 37; SEQ IDNOs:39 and 43; SEQ ID NOs:41 and 45; SEQ ID NOs:47 and 51; SEQ ID NOs:49and 53; SEQ ID NOs:55 and 59; SEQ ID NOs:57 and 61; SEQ ID NOs:63 and67; SEQ ID NOs:65 and 69; SEQ ID NOs:71 and 73; SEQ ID NOs:72 and 74;SEQ ID NOs:75 and 79; SEQ ID NOs:77 and 81; SEQ ID NOs:83 and 85; SEQ IDNOs:84 and 86; SEQ ID NOs:87 and 89; SEQ ID NOs:88 and 90; SEQ ID NOs:91and 93; SEQ ID NOs:92 and 94; SEQ ID NOs:95 and 97; and SEQ ID NOs:96and 98.

The first hinge of a TFc may comprise an aa sequence that differs in atmost 3, 2 or 1 aa deletions, additions or substitutions from an aasequence of a hinge provided herein, e.g., selected from the groupconsisting of SEQ ID NOs:4, 18, 19, 20, 21, 22, 263-265 and 267-273. Thefirst hinge of a TFc may comprise an aa sequence that is an aa sequenceselected from the group consisting of SEQ ID NOs:4, 18, 19, 20, 21, 22,263-265 and 267-273. The second hinge of a TFc may comprise an aasequence that differs in at most 3, 2 or 1 aa deletions, additions orsubstitutions from an aa sequence of a hinge provided herein, e.g.,selected from the group consisting of SEQ ID NOs:23, 24, 263-265 and267-273. The second hinge may comprise an aa sequence that is an aasequence selected from the group consisting of SEQ ID NOs:23, 24,263-265 and 267-273.

A CH2 domain of a TFc may comprise an aa sequence that is at least 70%,80%, 90%, 95%, 97%, 98%, or 99% identical to an aa sequence of a CH2domain provided herein, e.g., SEQ ID NO:25, 26, 261 or 262, or whichdiffers therefrom in at most 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 aadeletions, additions or substitutions.

The TFc may comprise in amino to carboxyl terminal order: a first hinge,a first CH2 domain, a first CH3 domain, a second hinge, a second CH2domain and a second CH3 domain, wherein (i) the first hinge comprises anaa sequence selected from the group consisting of SEQ ID NOs:4, 18, 19,263-265 and 267-273; (ii) the first CH2 domain is aglycosylated andcomprises the aa sequence set forth as SEQ ID NO:25; (iii) the first CH3domain comprises an aa sequence that is either sequence of a pair ofsequences selected from the group of pairs of CH3 domain sequencesconsisting of SEQ ID NOs:31 and 35; SEQ ID NOs:33 and 37; SEQ ID NOs:39and 43; SEQ ID NOs:41 and 45; SEQ ID NOs:47 and 51; SEQ ID NOs:49 and53; SEQ ID NOs:55 and 59; SEQ ID NOs:57 and 61; SEQ ID NOs:63 and 67;SEQ ID NOs:65 and 69; SEQ ID NOs:71 and 73; SEQ ID NOs:72 and 74; SEQ IDNOs:75 and 79; SEQ ID NOs:77 and 81; SEQ ID NOs:83 and 85; SEQ ID NOs:84and 86; SEQ ID NOs:87 and 89; SEQ ID NOs:88 and 90; SEQ ID NOs:91 and93; SEQ ID NOs:92 and 94; SEQ ID NOs:95 and 97; and SEQ ID NOs:96 and98; (iv) the second hinge comprises an aa sequence consisting of asequence selected from the group consisting of SEQ ID NO:23, 263-265 and267-273; (v) the second CH2 domain is aglycosylated and comprises the aasequence set forth in SEQ ID NO:25; and (vi) the second CH3 domaincomprises an aa sequence that is either sequence of a pair of sequencesselected from the group of pairs of CH3 domain sequences consisting ofSEQ ID NOs:31 and 35; SEQ ID NOs:33 and 37; SEQ ID NOs:39 and 43; SEQ IDNOs:41 and 45; SEQ ID NOs:47 and 51; SEQ ID NOs:49 and 53; SEQ ID NOs:55and 59; SEQ ID NOs:57 and 61; SEQ ID NOs:63 and 67; SEQ ID NOs:65 and69; SEQ ID NOs:71 and 73; SEQ ID NOs:72 and 74; SEQ ID NOs:75 and 79;SEQ ID NOs:77 and 81; SEQ ID NOs:83 and 85; SEQ ID NOs:84 and 86; SEQ IDNOs:87 and 89; SEQ ID NOs:88 and 90; SEQ ID NOs:91 and 93; SEQ ID NOs:92and 94; SEQ ID NOs:95 and 97; and SEQ ID NOs:96 and 98, wherein if thefirst CH3 domain comprises a first sequence of a pair of sequences, thesecond CH3 domain comprises the second sequence of the pair ofsequences; and if the first CH3 domain comprises the second sequence ofa pair of sequences, the second CH3 domain comprises the first sequenceof the pair of sequences.

A TFc may comprise in amino to carboxyl terminal order: a first hinge, afirst CH2 domain, a first CH3 domain, a second hinge, a second CH2domain and a second CH3 domain, wherein (i) the first hinge comprises anaa sequence selected from the group consisting of SEQ ID NOs:20, 21, 22,263-265 and 267-273; (ii) the first CH2 domain is aglycosylated andcomprises the aa sequence set forth in SEQ ID NO:26; (iii) the first CH3domain comprises an aa sequence that is either sequence of a pair ofsequences selected from the group of pairs of CH3 domain sequencesconsisting of SEQ ID NOs:31 and 35; SEQ ID NOs:33 and 37; SEQ ID NOs:39and 43; SEQ ID NOs:41 and 45; SEQ ID NOs:47 and 51; SEQ ID NOs:49 and53; SEQ ID NOs:55 and 59; SEQ ID NOs:57 and 61; SEQ ID NOs:63 and 67;SEQ ID NOs:65 and 69; SEQ ID NOs:71 and 73; SEQ ID NOs:72 and 74; SEQ IDNOs:75 and 79; SEQ ID NOs:77 and 81; SEQ ID NOs:83 and 85; SEQ ID NOs:84and 86; SEQ ID NOs:87 and 89; SEQ ID NOs:88 and 90; SEQ ID NOs:91 and93; SEQ ID NOs:92 and 94; SEQ ID NOs:95 and 97; and SEQ ID NOs:96 and98; (iv) the second hinge comprises an aa sequence consisting of SEQ IDNO:24, 263-265 and 267-273; (v) the second CH2 domain is aglycosylatedand comprises the aa sequence set forth in SEQ ID NO:26; and (vi) thesecond CH3 domain comprises an aa sequence that is either sequence of apair of sequences selected from the group of pairs of CH3 domainsequences consisting of SEQ ID NOs:31 and 35; SEQ ID NOs:33 and 37; SEQID NOs:39 and 43; SEQ ID NOs:41 and 45; SEQ ID NOs:47 and 51; SEQ IDNOs:49 and 53; SEQ ID NOs:55 and 59; SEQ ID NOs:57 and 61; SEQ ID NOs:63and 67; SEQ ID NOs:65 and 69; SEQ ID NOs:71 and 73; SEQ ID NOs:72 and74; SEQ ID NOs:75 and 79; SEQ ID NOs:77 and 81; SEQ ID NOs:83 and 85;SEQ ID NOs:84 and 86; SEQ ID NOs:87 and 89; SEQ ID NOs:88 and 90; SEQ IDNOs:91 and 93; SEQ ID NOs:92 and 94; SEQ ID NOs:95 and 97; and SEQ IDNOs:96 and 98, wherein if the first CH3 domain comprises a firstsequence of a pair of sequences, the second CH3 domain comprises thesecond sequence of the pair of sequences; and if the first CH3 domaincomprises the second sequence of a pair of sequences, the second CH3domain comprises the first sequence of the pair of sequences.

The first or the second Fc region of a TFc may comprise an aa sequencethat is at least 70%, 80%, 90%, 95%, 97%, 98%, or 99% identical to an aasequence of an Fc region provided herein, e.g., selected from the groupconsisting of SEQ ID NOs:99-166, or differs therefrom in at most 50, 40,30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 aa deletions, additions orsubstitutions. The first or the second Fc region comprises an aasequence selected from the group consisting of SEQ ID NOs:99-166. Thefirst and the second Fc region may comprise an aa sequence that is atleast 70%, 80%, 90%, 95%, 97%, 98%, or 99% identical to one aa sequenceof a pair of aa sequences selected from the group consisting of SEQ IDNOs:99 and 100; SEQ ID NOs:101 and 102; SEQ ID NOs:103 and 104; SEQ IDNOs:105 and 106; SEQ ID NOs:107 and 108; SEQ ID NOs:109 and 110; SEQ IDNOs:111 and 112; SEQ ID NOs:113 and 114; SEQ ID NOs:115 and 116; SEQ IDNOs:117 and 118; SEQ ID NOs:119 and 120; SEQ ID NOs:121 and 122; SEQ IDNOs:123 and 124; SEQ ID NOs:125 and 126; SEQ ID NOs:127 and 128; SEQ IDNOs:129 and 130; SEQ ID NOs:131 and 132; SEQ ID NOs:133 and 134; SEQ IDNOs:135 and 136; SEQ ID NOs:137 and 138; SEQ ID NOs:139 and 140; SEQ IDNOs:141 and 142; SEQ ID NOs:143 and 144; SEQ ID NOs:145 and 146; SEQ IDNOs:147 and 148; SEQ ID NOs:149 and 150; SEQ ID NOs:151 and 152; SEQ IDNOs:153 and 154; SEQ ID NOs:155 and 156; SEQ ID NOs:157 and 158; SEQ IDNOs:159 and 160; SEQ ID NOs:161 and 162; SEQ ID NOs:163 and 164; and SEQID NOs:165 and 166, or which differs therefrom in at most 50, 40, 30,25, 20, 15, 10, 5, 4, 3, 2, or 1 aa deletions, additions orsubstitutions, and wherein the first Fc region comprises a differentmember of the pair than is comprised by the second Fc region. The firstFc region and the second Fc region together may comprise a pair of twodifferent members, each member being an Fc aa sequence, wherein eachpair is selected from the group of pairs consisting of SEQ ID NOs:99 and100; SEQ ID NOs:101 and 102; SEQ ID NOs:103 and 104; SEQ ID NOs:105 and106; SEQ ID NOs:107 and 108; SEQ ID NOs:109 and 110; SEQ ID NOs:111 and112; SEQ ID NOs:113 and 114; SEQ ID NOs:115 and 116; SEQ ID NOs:117 and118; SEQ ID NOs:119 and 120; SEQ ID NOs:121 and 122; SEQ ID NOs:123 and124; SEQ ID NOs:125 and 126; SEQ ID NOs:127 and 128; SEQ ID NOs:129 and130; SEQ ID NOs:131 and 132; SEQ ID NOs:133 and 134; SEQ ID NOs:135 and136; SEQ ID NOs:137 and 138; SEQ ID NOs:139 and 140; SEQ ID NOs:141 and142; SEQ ID NOs:143 and 144; SEQ ID NOs:145 and 146; SEQ ID NOs:147 and148; SEQ ID NOs:149 and 150; SEQ ID NOs:151 and 152; SEQ ID NOs:153 and154; SEQ ID NOs:155 and 156; SEQ ID NOs:157 and 158; SEQ ID NOs:159 and160; SEQ ID NOs:161 and 162; SEQ ID NOs:163 and 164; and SEQ ID NOs:165and 166, each member aa sequence being at least 70%, 80%, 90%, 95%, 97%,98%, or 99% identical to, or differing in at most 50, 40, 30, 25, 20,15, 10, 5, 4, 3, 2, or 1 aa additions, deletions or substitutions fromeach sequence of each said pair, wherein the first Fc region comprises adifferent member of the pair than is comprised by the second Fc region.

A TFc may comprise an aa sequence that is at least 70%, 80%, 90%, 95%,97%, 98%, or 99% identical to an aa sequence of a TFc provided herein,e.g., selected from the group consisting of SEQ ID NOs:171, 173, 175,177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203,205, 207, 209, 211, 213, 215, 217, 219 and 221, or which differstherefrom in at most 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 aa additions,deletions or substitutions. The TFc may comprise an aa sequence selectedfrom the group consisting of SEQ ID NOs:171, 173, 175, 177, 179, 181,183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209,211, 213, 215, 217, 219 and 221.

A TFcA, e.g., a TFcBA, e.g., an anti-c-Met+anti-EGFR TFcBA or ananti-c-Kit+anti-RON TFcBA or an anti-FGFR2+anti-EPCAM TFcBA, maycomprise a heavy chain that comprises in amino to carboxyl terminalorder: a first heavy chain variable (VH) domain, a TFc, a connectinglinker and a second VH domain. The heavy chain may comprise in amino tocarboxyl terminal order: a first VH domain, a CH1 domain, a TFc, aconnecting linker and a second VH domain. The heavy chain may comprisein amino to carboxyl terminal order: a first VH domain, a CH1 domain, aTFc, a connecting linker, a second VH domain, an scFv linker and asecond light chain variable (VL) domain, wherein the second VH and VLdomains associate to form a second binding site. A TFcA may comprise alight chain that comprises a first VL domain that dimerizes with thefirst VH domain to form a first binding site. The light chain maycomprise a light chain constant (CL) domain that is linked to thecarboxyl terminus of the VL domain. The first binding site may be ananti-c-Met, anti-c-Kit, anti-ErbB2, anti-ErbB3, anti-ErbB4, anti-IGF1R,anti-IGF2R, anti-Insulin receptor, anti-RON, anti-EGFR, anti-VEGFR1,anti-VEGFR2, anti-TNFR, anti-FGFR1, anti-FGFR2, anti-FGFR3, anti-FGFR4,anti-PDGFR alpha, anti-PDGFR beta, anti-EPCAM or anti-EphA2 binding siteand the second binding site may be an anti-c-Met, anti-c-Kit,anti-ErbB2, anti-ErbB3, anti-ErbB4, anti-IGF1R, anti-IGF2R, anti-Insulinreceptor, anti-RON, anti-VEGFR1, anti-VEGFR2, anti-TNFR, anti-FGFR1,anti-FGFR2, anti-FGFR3, anti-FGFR4, anti-PDGFR alpha, anti-PDGFR beta,anti-EphA2 or anti-EGFR binding site. If a TFcA is a monovalent TFcA,the binding site may be an anti-c-Met, anti-c-Kit, anti-ErbB2,anti-ErbB3, anti-ErbB4, anti-IGF1R, anti-IGF2R, anti-Insulin receptor,anti-RON, anti-VEGFR1, anti-VEGFR2, anti-TNFR, anti-FGFR1, anti-FGFR2,anti-FGFR3, anti-FGFR4, anti-PDGFR alpha, anti-PDGFR beta, anti-EPCAM,anti-EphA2 or anti-EGFR binding site. An exemplary anti-c-Met bindingsite may comprise a VH domain comprising either or both of a) the aasequence of the VH Complementarity Determining Region (CDR)3 (VHCDR3) inSEQ ID NO:223 or 287 and b) a VLCDR3 comprising the aa sequence of theVLCDR3 in SEQ ID NO:231 or 289. Another exemplary the anti-c-Met bindingsite may comprise a VH domain comprising a set of three VH CDRscomprising VHCDR1, VCDR2 and VHCDR3, wherein VHCDR1, VHCDR2 and VHCDR3comprise the aa sequence of the VHCDR1, VHCDR2 and VHCDR3 in SEQ IDNO:223 or 231; and a VL domain comprising a set of three VLCDRscomprising VLCDR1, VLCDR2, and VLCDR3, wherein VLCDR1, VLCDR2 and VLCDR3comprise the aa sequence of the VLCDR1, VLCDR2 and VLCDR3 in SEQ IDNO:287 or 289, respectively. An exemplary anti-EGFR binding site maycomprise either or both of a) a VHCDR3 comprising the aa sequence of theVHCDR3 in SEQ ID NO:233, 237, 258, 275, 277 or 279 and b) a VLCDR3comprising the aa sequence of the VLCDR3 in SEQ ID NO:233, 237, 258,275, 277 or 279. An exemplary anti-EGFR binding site may comprise a VHdomain comprising a set of three VHCDRs comprising VHCDR1, VCDR2 andVHCDR3, wherein VHCDR1, VHCDR2 and VHCDR3 comprise the aa sequence ofthe VHCDR1, VHCDR2 and VHCDR3 in SEQ ID NO: 233, 237, 258, 275, 277 or279; and a VL domain comprising a set of three VLCDRs comprising VLCDR1,VLCDR2, and VLCDR3, wherein VLCDR1, VLCDR2 and VLCDR3 comprise the aasequence of the VLCDR1, VLCDR2 and VLCDR3 in SEQ ID NO:233, 237, 258,275, 277 or 279. The anti-c-Met, anti-c-Kit, anti-ErbB2, anti-ErbB3,anti-ErbB4, anti-IGF1R, anti-IGF2R, anti-Insulin receptor, anti-RON,anti-VEGFR1, anti-VEGFR2, anti-TNFR, anti-FGFR1, anti-FGFR2, anti-FGFR3,anti-FGFR4, anti-PDGFR alpha, anti-PDGFR beta, anti-EPCAM, anti-EphA2 oranti-EGFR binding site may comprise an N-terminal portion of the heavychain and an N-terminal portion of the light chain. The anti-EGFR,anti-c-Kit, anti-ErbB2, anti-ErbB3, anti-ErbB4, anti-IGF1R, anti-IGF2R,anti-Insulin receptor, anti-RON, anti-VEGFR1, anti-VEGFR2, anti-TNFR,anti-FGFR1, anti-FGFR2, anti-FGFR3, anti-FGFR4, anti-PDGFR alpha,anti-PDGFR beta, anti-EPCAM, anti-EphA2 or anti-c-Met binding site maybe comprised by a C-terminal scFv that is entirely comprised by theheavy chain to form a contiguous polypeptide.

An anti-c-Met binding site of a TFcA, e.g., a TFcBA, may be comprised byeither or both of a VH domain and a VL domain, wherein the VH domaincomprises an aa sequence that is at least 70%, 80%, 90%, 95%, 97%, 98%,or 99% identical to the VH domain of an anti-c-Met binding site, e.g.,set forth in SEQ ID NOs:223, 231, 287 or 289, or differs therefrom in atmost 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 aas deletions, additions orsubstitution; and the VL domain comprises an aa sequence that is atleast 70%, 80%, 90%, 95%, 97%, 98%, or 99% identical to the VL domain ofan anti-c-Met binding site provided herein, e.g., set forth in SEQ IDNOs:223, 231, 287 or 289, or differs therefrom in at most 10, 9, 8, 7,6, 5, 4, 3, 2, or 1 aas deletions, additions or substitution.

An anti-EGFR binding site of a TFcA, e.g., a TFcBA, may be comprised byeither or both of a VH domain and a VL domain, wherein the VH domaincomprises an aa sequence that is at least 70%, 80%, 90%, 95%, 97%, 98%,or 99% identical to the VH domain of an anti-EGFR binding site providedherein, e.g., set forth in SEQ ID NOs: 233, 237, 258, 275, 277 or 279,or differs therefrom in at most 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 aa(s)deletion(s), addition(s) or substitution(s); and the VL domain comprisesan aa sequence that is at least 70%, 80%, 90%, 95%, 97%, 98%, or 99%identical to the VL domain of an anti-EGFR binding site provided herein,e.g., set forth in SEQ ID NOs: 233, 237, 258, 275, 277 or 279, ordiffers therefrom in at most 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 aa(s)deletion(s), addition(s) or substitution(s).

A TFcA or TFcBA may be a charge-complementary paired TFcA or TFcBA,e.g., wherein: a charge-complementary paired TFcA or TFcBA is a TFcA orTFcBA that comprises a pair of charged amino acids comprising an aminoacid selected from group A and an amino acid selected from group B (acharge-complementary pair); wherein group A comprises all natural aminoacids with a pI of greater than 7 and group B comprises all naturalamino acids with a pI of less than 7, or optionally wherein group Acomprises His, Lys, and Arg, and group B comprises Asp, Glu, Asn, Phe,Gln, Tyr, Ser, Met, Thr, Ile, Gly, Val, Trp, Leu, Ala, and Pro; and saidcharge-complementary pair consists of a first amino acid residue and asecond amino acid residue, and said charge-complementary pair is aposition 297 charge-complementary pair or a position 299charge-complementary pair, wherein a position 297 charge-complementarypair is a charge-complementary pair with said first amino acid residuelocated at EU position 297 of said first Fc region and said second aminoacid residue located at EU position 297 of said second Fc region, and aposition 299 charge-complementary pair is a charge-complementary pairwith said first amino acid residue located at EU position 299 of saidfirst Fc region and said second amino acid residue located at EUposition 299 of said second Fc region. The charge-complementary pairedTFcA or TFcBA may comprise both a position 297 charge-complementary pairand a position 299 charge-complementary pair, wherein the first andsecond amino acid residues of the position 297 charge-complementary pairare the same as or different from the first and second amino acidresidues of the position 299 charge-complementary pair. Thecharge-complementary paired TFcA or may comprise a position 297charge-complementary pair and wherein the charge-complementary pairedTFcA or TFcBA is more stable than a TFcA or TFcBA that is not acharge-complementary paired TFcA or TFcBA but that is identical to thecharge-complementary paired TFcA or TFcBA except that amino acidresidues corresponding to the first and the second amino acid residuesare both residues consisting of the same charged amino acid, said samecharged amino acid being one of the amino acids of the position 297charge-complementary pair of the charge-complementary paired TFcA orTFcBA. The charge-complementary paired TFcA or TFcBA may comprise aposition 299 charge-complementary pair and wherein thecharge-complementary paired TFcA or TFcBA is more stable than a TFcA orTFcBA that is not a charge-complementary paired TFcA or TFcBA but thatis identical to the charge-complementary paired TFcA or TFcBA exceptthat amino acid residues corresponding to the first and the second aminoacid residues are both residues consisting of the same charged aminoacid, said same charged amino acid being one of the amino acids of theposition 299 charge-complementary pair of the charge-complementarypaired TFcA or TFcBA.

The first or the second binding site of a TFcA or TFcBA may bindspecifically to a human receptor protein selected from the groupconsisting of ErbB2, ErbB3, ErbB4, IGF1R, IGF2R, Insulin receptor, RON,c-Met, EGFR, VEGFR1, VEGFR2, TNFR, FGFR1-4, PDGFR (alpha and beta),c-Kit, EPCAM and EphA2.

Further provided herein are pharmaceutical compositions comprising aTFcA or TFcBA and a pharmaceutically acceptable carrier. Also providedare nucleic acid molecules, e.g., comprising at least one codingsequence, said at least one coding sequence encoding a heavy chain or alight chain of a TFcA or TFcBA. A nucleic acid molecule may comprise atleast two coding sequences, wherein one coding sequence encodes a heavychain of a TFcA or TFcBA and a second coding sequence encodes a lightchain of the TFcBA. Also provided are vectors, e.g., comprising one ormore nucleic acid molecules provided herein. Further provided are cells,e.g., host cells or isolated cells, comprising one or more vectorsand/or nucleic acid molecules provided herein. A cell may comprise anucleic acid molecule encoding the heavy chain of a TFcA or TFcBA and anucleic acid molecule encoding the light chain of the TFcA or TFcBA.

Also encompassed herein are methods of producing a TFcA or TFcBAcomprising culturing a host cell described herein under conditions inwhich the nucleic acids are expressed, and isolating the TFcA or TFcBA.A method for producing a TFcA or TFcBA may comprise culturing a celldescribed herein under conditions suitable for the expression of theTFcA or TFcBA.

Also provided herein are methods of treating a subject having cancer,said method comprising administering to a subject a therapeuticallyeffective amount of a TFcA or TFcBA, nucleic acid molecule, or vectordescribed herein.

Exemplary embodiments include but are not limited to the following itemsin connection with the subject matter which may be claimed.

-   1. An antibody, which is a Tandem Fc Bispecific Antibody (“TFcBA”),    wherein the TFcBA comprises a first binding site that is a single    anti-c-Met binding site and at least one second binding site that    specifically binds to a cell surface receptor other than c-Met;    optionally a cell surface receptor selected from ErbB2, ErbB3,    ErbB4, IGF1R, IGF2R, Insulin receptor, RON, EGFR, VEGFR1, VEGFR2,    TNFR, FGFR1, FGFR2, FGFR3, FGFR4, PDGFR alpha, PDGFR beta, c-Kit,    AXL, ALK, CEA, CD44, EPCAM and EphA2, wherein the anti-c-Met binding    site and the second binding site are linked through a Tandem Fc    (“TFc”); the TFc comprises a first Fc region and a second Fc region,    each said first Fc region and second Fc region having a C-terminus    and an N-terminus; the first Fc region and the second Fc region are    linked through a TFc linker having a C-terminus and an N-terminus to    form a contiguous polypeptide; and

the first and the second Fc regions associate to form an Fc dimer.

-   2. The TFcBA of embodiment 1, wherein    -   a. the TFcBA inhibits signal transduction induced by either or        both of HGF and a cognate ligand of the receptor specifically        bound by the at least one second binding site with an IC50 of 10        nM or less or 1 nM or less or 100 pM or less, or a maximal        percent inhibition of at least 70% or at least 80% or at least        90%, as indicated by inhibition of phosphorylation of either or        both of c-Met and the receptor specifically bound by the at        least one second binding site; or    -   b. expression of the TFcBA in a cell produces (i) more correctly        formed TFcAB molecules relative to the expression of a        multivalent antibody that does not comprise a TFc or (ii) more        than 80% of correctly formed TFcAB molecules as determined by        Size Exclusion Chromatography (SEC).-   3. The TFcBA of embodiment 1 or 2, wherein the first Fc region and    the second Fc region comprise a first and a second CH3 domain,    respectively, each said CH3 domain having a C-terminus and an    N-terminus-   4. The TFcBA of anyone of embodiments 1-3, wherein the first and the    second Fc regions comprise a first and a second CH2 domain,    respectively, each said CH2 domain having a C-terminus and an    N-terminus-   5. The TFcBA of anyone of embodiments 1-4, wherein the first and the    second Fc regions comprise a first and a second hinge, respectively,    each said first hinge and said second hinge having a C-terminus and    an N-terminus-   6. The TFcBA of any one of embodiments 1-5, wherein the second hinge    does not comprise an upper hinge subdomain-   7. The TFcBA of embodiment 6, wherein the TFc comprised in the TFcBA    comprises in amino to carboxyl terminal order: a first CH2 domain, a    first CH3 domain, a TFc linker, a second CH2 domain and a second CH3    domain-   8. The TFcBA of embodiment 6, wherein the TFc comprised in the TFcBA    comprises in amino to carboxyl terminal order: a first hinge, a    first CH2 domain, a first CH3 domain, a TFc linker, a second CH2    domain and a second CH3 domain.-   9. The TFcBA of embodiment 6, wherein the TFc comprised in the TFcBA    comprises in amino to carboxyl terminal order: a first hinge, a    first CH2 domain, a first CH3 domain, a TFc linker, a second hinge,    a second CH2 domain and a second CH3 domain.-   10. The TFcBA of embodiment 9, wherein the first hinge comprises an    upper hinge subdomain, a core hinge subdomain and a lower hinge    subdomain and the second hinge comprises a core hinge subdomain and    a lower hinge subdomain, but not an upper hinge subdomain, each said    hinge sub-domain having a C-terminus and an N-terminus-   11. A TFcBA of any one of embodiments 1-10, wherein the TFc    comprised by the TFcBA comprises in amino to carboxyl terminal    order: a first hinge, which is linked at its C-terminus to the    N-terminus of a first CH2 domain, which is linked at its C-terminus    to the N-terminus of a first CH3 domain, which is linked at its    C-terminus to the N-terminus of a TFc linker, which is linked at its    C-terminus to the N-terminus of a second hinge, which is linked at    its C-terminus to the N-terminus of a second CH2 domain, which is    linked at its C-terminus to the N-terminus of a second CH3 domain-   12. The TFcBA of any one of embodiments 1-11, wherein the TFc linker    comprises 20-50 aas.-   13. The TFcBA of embodiment 12, wherein the TFc linker is a Gly-Ser    linker-   14. The TFcBA of embodiment 13, wherein the TFc linker comprises    (Gly₄Ser)_(n), wherein n is 4, 5, 6, 7 or 8.-   15. The TFcBA of any one of embodiments 1-14, wherein the TFc is an    IgG1 TFc.-   16. The TFcBA of any of embodiments 1-14, wherein the TFc is a    hybrid TFc.-   17. The TFcBA of embodiment 16, wherein the TFc is an IgG1/IgG4 TFc.-   18. The TFcBA of embodiment 15, wherein the TFc comprises in amino    to carboxyl terminal order: a first IgG1 hinge, a first IgG1 CH2    domain, a first IgG1 CH3 domain, a TFc linker, a second IgG1 hinge,    a second IgG1 CH2 domain, and a second IgG1 CH3 domain-   19. The TFcBA of embodiment 17, wherein the hybrid TFc comprises in    amino to carboxyl terminal order: a first IgG1/IgG4 hinge, a first    IgG4 CH2 domain, a first IgG1 CH3 domain, a TFc linker, a second    IgG4 hinge, a second IgG4 CH2 domain, and a second IgG1 CH3 domain-   20. The TFcBA of anyone of embodiments 1-19, wherein either or both    of the first CH3 domain and the second CH3 domain comprise one or    more aa modifications that enhance or stabilize the binding between    the first and the second Fc regions.-   21. The TFcBA of embodiment 20, wherein each of the first CH3 domain    and the second CH3 domain comprises an amino acid modification,    which modification is an Association Enhancing Modification (“AEM”)    that enhances the association of the first CH3 domain with the    second CH3 domain.-   22. The TFcBA of embodiment 21, wherein the AEM is comprised by a    module selected from the group consisting of AEM module 1, AEM    module 2, AEM module 3 and AEM module 4.-   23. The TFcBA of anyone of embodiments 1-22, wherein either or both    of the first Fc region and the second Fc region comprises an aa    modification that adds a cysteine as an insertion or replacement,    which cysteine forms a disulfide bond with a cysteine in the other    Fc region (a “DiS” modification).-   24. The TFcBA of embodiment 23, wherein either or both of the first    and the second Fc region comprise a DiS modification in a hinge.-   25. The TFcBA of embodiment 23, wherein either or both of the first    and the second Fc region comprise a DiS modification in a CH3 domain-   26. The TFcBA of any one of embodiments 23-25, wherein the DiS    modification is comprised by DiS module 1 or DiS module 2.-   27. The TFcBA of any one of embodiments 1-26, wherein each of the    first CH3 domain and the second CH3 domain comprises one or more AEM    modifications and one or more DiS modifications.-   28. The TFcBA of any one of embodiments 1-27, wherein either or both    of the first and the second CH3 domains comprises an aa sequence    that is at least 70% identical to an aa sequence selected from the    group consisting of SEQ ID NOs:27-98, or which differs therefrom in    at most 30 aa additions, deletions or substitutions.-   29. The TFcBA of embodiment 28, wherein the first CH3 domain or the    second CH3 domain comprises an aa sequence selected from the group    consisting of SEQ ID NOs:27-98.-   30. The TFcBA of any one of embodiments 1-28, wherein the first CH3    and second CH3 domains together comprise a pair of two different    members, each member being a CH3 aa sequence, each pair selected    from the group of pairs consisting of SEQ ID NOs:31 and 35; SEQ ID    NOs:33 and 37; SEQ ID NOs:39 and 43; SEQ ID NOs:41 and 45; SEQ ID    NOs:47 and 51; SEQ ID NOs:49 and 53; SEQ ID NOs:55 and 59; SEQ ID    NOs:57 and 61; SEQ ID NOs:63 and 67; SEQ ID NOs:65 and 69; SEQ ID    NOs:71 and 73; SEQ ID NOs:72 and 74; SEQ ID NOs:75 and 79; SEQ ID    NOs:77 and 81; SEQ ID NOs:83 and 85; SEQ ID NOs:84 and 86; SEQ ID    NOs:87 and 89; SEQ ID NOs:88 and 90; SEQ ID NOs:91 and 93; SEQ ID    NOs:92 and 94; SEQ ID NOs:95 and 97; and SEQ ID NOs:96 and 98, each    member aa sequence being at least 70% identical to, or differing in    at most 30 aa additions, deletions or substitutions from the each    sequence of each said pair, wherein the first CH3 domain comprises a    different member of the pair than is comprised by the second CH3    domain-   31. The TFcBA of embodiment 30, wherein the first and the second CH3    domains each comprise an aa sequence that identical to an aa    sequence of a member of the pair of CH3 aa sequences selected from    the group consisting of SEQ ID NOs:31 and 35; SEQ ID NOs:33 and 37;    SEQ ID NOs:39 and 43; SEQ ID NOs:41 and 45; SEQ ID NOs:47 and 51;    SEQ ID NOs:49 and 53; SEQ ID NOs:55 and 59; SEQ ID NOs:57 and 61;    SEQ ID NOs:63 and 67; SEQ ID NOs:65 and 69; SEQ ID NOs:71 and 73;    SEQ ID NOs:72 and 74; SEQ ID NOs:75 and 79; SEQ ID NOs:77 and 81;    SEQ ID NOs:83 and 85; SEQ ID NOs:84 and 86; SEQ ID NOs:87 and 89;    SEQ ID NOs:88 and 90; SEQ ID NOs:91 and 93; SEQ ID NOs:92 and 94;    SEQ ID NOs:95 and 97; and SEQ ID NOs:96 and 98.-   32. The TFcBA of any one of embodiments 1-31, wherein the first    hinge comprises an aa sequence that differs in at most 3 aa    deletions, additions or substitutions from an aa sequence selected    from the group consisting of SEQ ID NOs:4, 18, 19, 20, 21, 22,    263-265 and 267-273.-   33. The TFcBA of embodiment 32, wherein the first hinge comprises an    aa sequence that is an aa sequence selected from the group    consisting of SEQ ID NOs:4, 18, 19, 20, 21, 22, 263-265 and 267-273.-   34. The TFcBA of any one of embodiments 1-33, wherein the second    hinge comprises an aa sequence that differs in at most 3 aa    deletions, additions or substitutions from an aa sequence selected    from the group consisting of SEQ ID NOs:23, 24, 263-265 and 267-273.-   35. The TFcBA of embodiment 34, wherein the second hinge comprises    an aa sequence that is an aa sequence selected from the group    consisting of SEQ ID NOs:23, 24, 263-265 and 267-273.-   36. The TFcBA of any one of embodiments 1-35, comprising a CH2    domain comprising an aa sequence that is at least 70% identical to    SEQ ID NO:25, 26, 261 or 262, or which differs therefrom in at most    30 aa deletions, additions or substitutions.-   37. The TFcBA of any one of embodiments 1-36, wherein the TFc    comprises in amino to carboxyl terminal order: a first hinge, a    first CH2 domain, a first CH3 domain, a second hinge, a second CH2    domain and a second CH3 domain, wherein    -   a. the first hinge comprises an aa sequence selected from the        group consisting of SEQ ID NOs:4, 18, 19, 263-265 and 267-273;    -   b. the first CH2 domain is aglycosylated and comprises the aa        sequence set forth as SEQ ID NO:25;    -   c. the first CH3 domain comprises an aa sequence that is either        sequence of a pair of sequences selected from the group of pairs        of CH3 domain sequences consisting of SEQ ID NOs:31 and 35; SEQ        ID NOs:33 and 37; SEQ ID NOs:39 and 43; SEQ ID NOs:41 and 45;        SEQ ID NOs:47 and 51; SEQ ID NOs:49 and 53; SEQ ID NOs:55 and        59; SEQ ID NOs:57 and 61; SEQ ID NOs:63 and 67; SEQ ID NOs:65        and 69; SEQ ID NOs:71 and 73; SEQ ID NOs:72 and 74; SEQ ID        NOs:75 and 79; SEQ ID NOs:77 and 81; SEQ ID NOs:83 and 85; SEQ        ID NOs:84 and 86; SEQ ID NOs:87 and 89; SEQ ID NOs:88 and 90;        SEQ ID NOs:91 and 93; SEQ ID NOs:92 and 94; SEQ ID NOs:95 and        97; and SEQ ID NOs:96 and 98;    -   d. the second hinge comprises an aa sequence consisting of a        sequence selected from the group consisting of SEQ ID NO:23,        263-265 and 267-273;    -   e. the second CH2 domain is aglycosylated and comprises the aa        sequence set forth in SEQ ID NO:25; and    -   f. the second CH3 domain comprises an aa sequence that is either        sequence of a pair of sequences selected from the group of pairs        of CH3 domain sequences consisting of SEQ ID NOs:31 and 35; SEQ        ID NOs:33 and 37; SEQ ID NOs:39 and 43; SEQ ID NOs:41 and 45;        SEQ ID NOs:47 and 51; SEQ ID NOs:49 and 53; SEQ ID NOs:55 and        59; SEQ ID NOs:57 and 61; SEQ ID NOs:63 and 67; SEQ ID NOs:65        and 69; SEQ ID NOs:71 and 73; SEQ ID NOs:72 and 74; SEQ ID        NOs:75 and 79; SEQ ID NOs:77 and 81; SEQ ID NOs:83 and 85; SEQ        ID NOs:84 and 86; SEQ ID NOs:87 and 89; SEQ ID NOs:88 and 90;        SEQ ID NOs:91 and 93; SEQ ID NOs:92 and 94; SEQ ID NOs:95 and        97; and SEQ ID NOs:96 and 98, wherein if the first CH3 domain        comprises a first sequence of a pair of sequences, the second        CH3 domain comprises the second sequence of the pair of        sequences; and if the first CH3 domain comprises the second        sequence of a pair of sequences, the second CH3 domain comprises        the first sequence of the pair of sequences.-   38. The TFcBA of any one of embodiments 1-36, wherein the TFc    comprises in amino to carboxyl terminal order: a first hinge, a    first CH2 domain, a first CH3 domain, a second hinge, a second CH2    domain and a second CH3 domain, wherein    -   a. the first hinge comprises an aa sequence selected from the        group consisting of SEQ ID NOs:20, 21, 22, 263-265 and 267-273;    -   b. the first CH2 domain is aglycosylated and comprises the aa        sequence set forth in SEQ ID NO:26;    -   c. the first CH3 domain comprises an aa sequence that is either        sequence of a pair of sequences selected from the group of pairs        of CH3 domain sequences consisting of SEQ ID NOs:31 and 35; SEQ        ID NOs:33 and 37; SEQ ID NOs:39 and 43; SEQ ID NOs:41 and 45;        SEQ ID NOs:47 and 51; SEQ ID NOs:49 and 53; SEQ ID NOs:55 and        59; SEQ ID NOs:57 and 61; SEQ ID NOs:63 and 67; SEQ ID NOs:65        and 69; SEQ ID NOs:71 and 73; SEQ ID NOs:72 and 74; SEQ ID        NOs:75 and 79; SEQ ID NOs:77 and 81; SEQ ID NOs:83 and 85; SEQ        ID NOs:84 and 86; SEQ ID NOs:87 and 89; SEQ ID NOs:88 and 90;        SEQ ID NOs:91 and 93; SEQ ID NOs:92 and 94; SEQ ID NOs:95 and        97; and SEQ ID NOs:96 and 98;    -   d. the second hinge comprises an aa sequence consisting of SEQ        ID NO:24, 263-265 and 267-273;    -   e. the second CH2 domain is aglycosylated and comprises the aa        sequence set forth in SEQ ID NO:26; and    -   f. the second CH3 domain comprises an aa sequence that is either        sequence of a pair of sequences selected from the group of pairs        of CH3 domain sequences consisting of SEQ ID NOs:31 and 35; SEQ        ID NOs:33 and 37; SEQ ID NOs:39 and 43; SEQ ID NOs:41 and 45;        SEQ ID NOs:47 and 51; SEQ ID NOs:49 and 53; SEQ ID NOs:55 and        59; SEQ ID NOs:57 and 61; SEQ ID NOs:63 and 67; SEQ ID NOs:65        and 69; SEQ ID NOs:71 and 73; SEQ ID NOs:72 and 74; SEQ ID        NOs:75 and 79; SEQ ID NOs:77 and 81; SEQ ID NOs:83 and 85; SEQ        ID NOs:84 and 86; SEQ ID NOs:87 and 89; SEQ ID NOs:88 and 90;        SEQ ID NOs:91 and 93; SEQ ID NOs:92 and 94; SEQ ID NOs:95 and        97; and SEQ ID NOs:96 and 98, wherein if the first CH3 domain        comprises a first sequence of a pair of sequences, the second        CH3 domain comprises the second sequence of the pair of        sequences; and if the first CH3 domain comprises the second        sequence of a pair of sequences, the second CH3 domain comprises        the first sequence of the pair of sequences.-   39. The TFcBA of any one of embodiments 1-38, wherein the first or    the second Fc region comprises an aa sequence that is at least 70%    identical to an aa sequence selected from the group consisting of    SEQ ID NOs:99-166, or differs therefrom in at most 50 aa deletions,    additions or substitutions.-   40. The TFcBA of any one of embodiment 39, wherein the first or the    second Fc region comprises an aa sequence selected from the group    consisting of SEQ ID NOs:99-166.-   41. The TFcBA of embodiment 39, wherein either or both of the first    and the second Fc region comprises an aa sequence that is at least    70% identical to one aa sequence of a pair of aa sequences selected    from the group consisting of SEQ ID NOs:99 and 100; SEQ ID NOs:101    and 102; SEQ ID NOs:103 and 104; SEQ ID NOs:105 and 106; SEQ ID    NOs:107 and 108; SEQ ID NOs:109 and 110; SEQ ID NOs:111 and 112; SEQ    ID NOs:113 and 114; SEQ ID NOs:115 and 116; SEQ ID NOs:117 and 118;    SEQ ID NOs:119 and 120; SEQ ID NOs:121 and 122; SEQ ID NOs:123 and    124; SEQ ID NOs:125 and 126; SEQ ID NOs:127 and 128; SEQ ID NOs:129    and 130; SEQ ID NOs:131 and 132; SEQ ID NOs:133 and 134; SEQ ID    NOs:135 and 136; SEQ ID NOs:137 and 138; SEQ ID NOs:139 and 140; SEQ    ID NOs:141 and 142; SEQ ID NOs:143 and 144; SEQ ID NOs:145 and 146;    SEQ ID NOs:147 and 148; SEQ ID NOs:149 and 150; SEQ ID NOs:151 and    152; SEQ ID NOs:153 and 154; SEQ ID NOs:155 and 156; SEQ ID NOs:157    and 158; SEQ ID NOs:159 and 160; SEQ ID NOs:161 and 162; SEQ ID    NOs:163 and 164; and SEQ ID NOs:165 and 166, or which differs    therefrom in at most 50 aa deletions, additions or substitutions,    and wherein the first Fc region comprises a different member of the    pair than is comprised by the second Fc region.-   42. The TFcBA of embodiment 40, wherein the first Fc region and the    second Fc region together comprise a pair of two different members,    each member being an Fc aa sequence, wherein each pair is selected    from the group of pairs consisting of SEQ ID NOs:99 and 100; SEQ ID    NOs:101 and 102; SEQ ID NOs:103 and 104; SEQ ID NOs:105 and 106; SEQ    ID NOs:107 and 108; SEQ ID NOs:109 and 110; SEQ ID NOs:111 and 112;    SEQ ID NOs:113 and 114; SEQ ID NOs:115 and 116; SEQ ID NOs:117 and    118; SEQ ID NOs:119 and 120; SEQ ID NOs:121 and 122; SEQ ID NOs:123    and 124; SEQ ID NOs:125 and 126; SEQ ID NOs:127 and 128; SEQ ID    NOs:129 and 130; SEQ ID NOs:131 and 132; SEQ ID NOs:133 and 134; SEQ    ID NOs:135 and 136; SEQ ID NOs:137 and 138; SEQ ID NOs:139 and 140;    SEQ ID NOs:141 and 142; SEQ ID NOs:143 and 144; SEQ ID NOs:145 and    146; SEQ ID NOs:147 and 148; SEQ ID NOs:149 and 150; SEQ ID NOs:151    and 152; SEQ ID NOs:153 and 154; SEQ ID NOs:155 and 156; SEQ ID    NOs:157 and 158; SEQ ID NOs:159 and 160; SEQ ID NOs:161 and 162; SEQ    ID NOs:163 and 164; and SEQ ID NOs:165 and 166, each member aa    sequence being at least 70% identical to, or differing in at most 30    aa additions, deletions or substitutions from each sequence of each    said pair, wherein the first Fc region comprises a different member    of the pair than is comprised by the second Fc region.-   43. The TFcBA of any one of embodiments 1-42, comprising a TFc    comprising an aa sequence that is at least 70% identical to an aa    sequence selected from the group consisting of SEQ ID NOs:171, 173,    175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199,    201, 203, 205, 207, 209, 211, 213, 215, 217, 219 and 221 or which    differs therefrom in at most 30 aa additions, deletions or    substitutions.-   44. The TFcBA of embodiment 43, comprising a TFc comprising an aa    sequence selected from the group consisting of SEQ ID NOs:171, 173,    175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199,    201, 203, 205, 207, 209, 211, 213, 215, 217, 219 and 221.-   45. The TFcBA of any one of embodiments 1-44, comprising a heavy    chain that comprises in amino to carboxyl terminal order: a first    heavy chain variable (VH) domain, a TFc, a connecting linker and a    second VH domain-   46. The TFcBA of embodiment 45, wherein the heavy chain comprises in    amino to carboxyl terminal order: a first VH domain, a CH1 domain, a    TFc, a connecting linker and a second VH domain-   47. The TFcBA of embodiment 46, wherein the heavy chain comprises in    amino to carboxyl terminal order: a first VH domain, a CH1 domain, a    TFc, a connecting linker, a second VH domain, an scFv linker and a    second light chain variable (VL) domain, wherein the second VH and    VL domains associate to form a second binding site.-   48. The TFcBA of embodiment 47, comprising a light chain that    comprises a first VL domain that dimerizes with the first VH domain    to form a first binding site.-   49. The TFcBA of embodiment 48, wherein the light chain comprises a    light chain constant (CL) domain that is linked to the carboxyl    terminus of the VL domain-   50. The TFcBA of any one of embodiments 1-49, wherein the first    binding site is an N-terminal binding site and the second binding    site is a C-terminal binding site.-   51. The TFcBA of any one of embodiments 1-50, wherein the anti-c-Met    binding site comprises a VH comprising either or both of a) the aa    sequence of the VH Complementarity Determining Region (CDR)3    (VHCDR3) in SEQ ID NO:223 or 287 and b) a VLCDR3 comprising the aa    sequence of the VLCDR3 in SEQ ID NO:231 or 289.-   52. The TFcBA of any one of embodiments 1-51, wherein the anti-c-Met    binding site comprises a VH domain comprising a set of three VH    Complementarity Determining Regions (CDRs) comprising VHCDR1, VCDR2    and VHCDR3, wherein VHCDR1, VHCDR2 and VHCDR3 comprise the aa    sequence of the VHCDR1, VHCDR2 and VHCDR3 in SEQ ID NO:223 or 231;    and a VL domain comprising a set of three VLCDRs comprising VLCDR1,    VLCDR2, and VLCDR3, wherein VLCDR1, VLCDR2 and VLCDR3 comprise the    aa sequence of the VLCDR1, VLCDR2 and VLCDR3 in SEQ ID NO:287 or    289, respectively.-   53. The TFcBA of any one of embodiments 1-52, wherein the second    binding site is an anti-EGFR binding site that comprises either or    both of a) a VHCDR3 comprising the aa sequence of the VHCDR3 in SEQ    ID NO:233, 237, 258, 275, 277 or 279 and b) a VLCDR3 comprising the    aa sequence of the VLCDR3 in SEQ ID NO:233, 237, 258, 275, 277 or    279.-   54. The TFcBA of any one of embodiments 1-53, wherein the second    binding site is an anti-EGFR binding site that comprises a VH domain    comprising a set of three VHCDRs comprising VHCDR1, VCDR2 and    VHCDR3, wherein VHCDR1, VHCDR2 and VHCDR3 comprise the aa sequence    of the VHCDR1, VHCDR2 and VHCDR3 in SEQ ID NO: 233, 237, 258, 275,    277 or 279; and a VL domain comprising a set of three VLCDRs    comprising VLCDR1, VLCDR2, and VLCDR3, wherein VLCDR1, VLCDR2 and    VLCDR3 comprise the aa sequence of the VLCDR1, VLCDR2 and VLCDR3 in    SEQ ID NO:233, 237, 258, 275, 277 or 279.-   55. The TFcBA of any one of embodiments 1-54, wherein the anti-c-Met    binding site comprises an N-terminal portion of the heavy chain and    an N-terminal portion of the light chain.-   56. The TFcBA of any one of embodiments 1-55, wherein the second    binding site is comprised by a C-terminal scFv that is entirely    comprised by the heavy chain.-   57. The TFcBA of any one of embodiments 1-56, wherein the anti-c-Met    binding site is comprised by either or both of a VH domain and a VL    domain, wherein the VH domain comprises an aa sequence that is at    least 70% identical to the VH domain set forth in SEQ ID NOs:223,    231, 287 or 289 or differs therefrom in at most 10 aas deletions,    additions or substitution; and the VL domain comprises an aa    sequence that is at least 70% identical to the VL domain set forth    in SEQ ID NOs:223, 231, 287 or 289 or differs therefrom in at most    10 aas deletions, additions or substitution.-   58. The TFcBA of any one of embodiments 1-57, wherein the second    binding site is an anti-EGFR binding site that is comprised by    either or both of a VH domain and a VL domain, wherein the VH domain    comprises an aa sequence that is at least 70% identical to the VH    domain set forth in SEQ ID NOs: 233, 237, 258, 275, 277 or 279 or    differs therefrom in at most 10 aas deletions, additions or    substitution; and the VL domain comprises an aa sequence that is at    least 70% identical to the VL domain set forth in SEQ ID NOs: 233,    237, 258, 275, 277 or 279 or differs therefrom in at most 10 aas    deletions, additions or substitutions.-   59. An Ab which is a TFcBA, wherein the TFcBA comprises a first    binding site and a second binding site, wherein the first binding    site binds to a first target and the second binding site binds to a    second target, and wherein    -   the first and the second binding sites are linked through a TFc;    -   the TFc comprises a first Fc region and a second Fc region, each        said first Fc region and second Fc region having a C-terminus        and an N-terminus; the first Fc region and the second Fc region        are linked through a TFc linker having a C-terminus and an        N-terminus to form a contiguous polypeptide;    -   the first and the second Fc regions associate to form an Fc        dimer; and    -   either or both of the first and the second Fc region comprise        one or more aa modification to enhance or stabilize the binding        between the first and the second Fc region.-   60. The TFcBA of embodiment 59, wherein    -   a. the TFcBA inhibits signal transduction through either or both        of the first and the second target; or    -   b. expression of the TFcBA in a cell produces (i) more correctly        formed TFcAB molecules relative to the expression of a        multivalent antibody that does not comprise a TFc or (ii) more        than 80% of correctly formed TFcAB molecules as determined by        Size Exclusion Chromatography (SEC).-   61. The TFcBA of embodiment 59 or 60, wherein the first Fc region    and the second Fc region comprise a first and a second CH3 domain,    respectively, each said CH3 domain having a C-terminus and an    N-terminus-   62. The TFcBA of anyone of embodiments 59-61, wherein the first and    the second Fc regions comprise a first and a second CH2 domain,    respectively, each said CH2 domain having a C-terminus and an    N-terminus-   63. The TFcBA of anyone of embodiments 59-62, wherein the first and    the second Fc regions comprise a first and a second hinge,    respectively, each said first hinge and said second hinge having a    C-terminus and an N-terminus-   64. The TFcBA of anyone of embodiments 59-63, wherein the second    hinge does not comprise an upper hinge subdomain-   65. The TFcBA of embodiment 64, wherein the TFc comprised in the    TFcBA comprises in amino to carboxyl terminal order: a first CH2    domain, a first CH3 domain, a TFc linker, a second CH2 domain and a    second CH3 domain-   66. The TFcBA of embodiment 64, wherein the TFc comprised in the    TFcBA comprises in amino to carboxyl terminal order: a first hinge,    a first CH2 domain, a first CH3 domain, a TFc linker, a second CH2    domain and a second CH3 domain.-   67. The TFcBA of embodiment 64, wherein the TFc comprised in the    TFcBA comprises in amino to carboxyl terminal order: a first hinge,    a first CH2 domain, a first CH3 domain, a TFc linker, a second    hinge, a second CH2 domain and a second CH3 domain.-   68. The TFcBA of embodiment 67, wherein the first hinge comprises an    upper hinge subdomain, a core hinge subdomain and a lower hinge    subdomain and the second hinge comprises a core hinge subdomain and    a lower hinge subdomain, but not an upper hinge subdomain, each said    hinge sub-domain having a C-terminus and an N-terminus-   69. A TFcBA of any one of embodiments 59-68, wherein the TFc    comprised by the TFcBA comprises in amino to carboxyl terminal    order: a first hinge, which is linked at its C-terminus to the    N-terminus of a first CH2 domain, which is linked at its C-terminus    to the N-terminus of a first CH3 domain, which is linked at its    C-terminus to the N-terminus of a TFc linker, which is linked at its    C-terminus to the N-terminus of a second hinge, which is linked at    its C-terminus to the N-terminus of a second CH2 domain, which is    linked at its C-terminus to the N-terminus of a second CH3 domain-   70. The TFcBA of any one of embodiments 59-69, wherein the TFc    linker comprises 20-50 aas.-   71. The TFcBA of embodiment 70, wherein the TFc linker is a Gly-Ser    linker-   72. The TFcBA of embodiment 71, wherein the TFc linker comprises    (Gly₄Ser)_(n), wherein n is 4, 5, 6, 7 or 8.-   73. The TFcBA of any one of embodiments 59-72, wherein the TFc is an    IgG1 TFc.-   74. The TFcBA of any of embodiments 59-72, wherein the TFc is a    hybrid TFc.-   75. The TFcBA of embodiment 74, wherein the TFc is an IgG1/IgG4 TFc.-   76. The TFcBA of embodiment 73, wherein the TFc comprises in amino    to carboxyl terminal order: a first IgG1 hinge, a first IgG1 CH2    domain, a first IgG1 CH3 domain, a TFc linker, a second IgG1 hinge,    a second IgG1 CH2 domain, and a second IgG1 CH3 domain-   77. The TFcBA of embodiment 75, wherein the hybrid TFc comprises in    amino to carboxyl terminal order: a first IgG1/IgG4 hinge, a first    IgG4 CH2 domain, a first IgG1 CH3 domain, a TFc linker, a second    IgG4 hinge, a second IgG4 CH2 domain, and a second IgG1 CH3 domain-   78. The TFcBA of anyone of embodiments 59-77, wherein either or both    of the first CH3 domain and the second CH3 domain comprise one or    more aa modifications that enhance or stabilize the binding between    the first and the second Fc regions.-   79. The TFcBA of embodiment 78, wherein each of the first CH3 domain    and the second CH3 domain comprises an amino acid modification,    which modification is an Association Enhancing Modification (“AEM”)    that enhances the association of the first CH3 domain with the    second CH3 domain.-   80. The TFcBA of embodiment 79, wherein the AEM is comprised by a    module selected from the group consisting of AEM module 1, AEM    module 2, AEM module 3 and AEM module 4.-   81. The TFcBA of anyone of embodiments 1-80, wherein either or both    of the first Fc region and the second Fc region comprises an aa    modification that adds a cysteine as an insertion or replacement,    which cysteine forms a disulfide bond with a cysteine in the other    Fc region (a “DiS” modification).-   82. The TFcBA of embodiment 81, wherein either or both of the first    Fc region and the second Fc region comprise a DiS modification in a    hinge.-   83. The TFcBA of embodiment 81, wherein either or both of the first    Fc region and the second Fc region comprise a DiS modification in a    CH3 domain-   84. The TFcBA of any one of embodiments 80-83, wherein the DiS    modification is comprised by DiS module 1 or DiS module 2.-   85. The TFcBA of any one of embodiments 59-84, wherein each of the    first CH3 domain and the second CH3 domain comprises one or more AEM    modifications and one or more DiS modifications.-   86. The TFcBA of any one of embodiments 1-85, wherein either or both    of the first and the second CH3 domains comprises an aa sequence    that is at least 70% identical to an aa sequence selected from the    group consisting of SEQ ID NOs:27-98, or which differs therefrom in    at most 30 aa additions, deletions or substitutions.-   87. The TFcBA of embodiment 28, wherein the first CH3 domain or the    second CH3 domain comprises an aa sequence selected from the group    consisting of SEQ ID NOs:27-98.-   88. The TFcBA of any one of embodiments 1-86, wherein the first CH3    and second CH3 domains together comprise a pair of two different    members, each member being a CH3 aa sequence, each pair selected    from the group of pairs consisting of SEQ ID NOs:31 and 35; SEQ ID    NOs:33 and 37; SEQ ID NOs:39 and 43; SEQ ID NOs:41 and 45; SEQ ID    NOs:47 and 51; SEQ ID NOs:49 and 53; SEQ ID NOs:55 and 59; SEQ ID    NOs:57 and 61; SEQ ID NOs:63 and 67; SEQ ID NOs:65 and 69; SEQ ID    NOs:71 and 73; SEQ ID NOs:72 and 74; SEQ ID NOs:75 and 79; SEQ ID    NOs:77 and 81; SEQ ID NOs:83 and 85; SEQ ID NOs:84 and 86; SEQ ID    NOs:87 and 89; SEQ ID NOs:88 and 90; SEQ ID NOs:91 and 93; SEQ ID    NOs:92 and 94; SEQ ID NOs:95 and 97; and SEQ ID NOs:96 and 98, each    member aa sequence being at least 70% identical to, or differing in    at most 30 aa additions, deletions or substitutions from the each    sequence of each said pair, wherein the first CH3 domain comprises a    different member of the pair than is comprised by the second CH3    domain-   89. The TFcBA of embodiment 88, wherein the first and the second CH3    domains each comprise an aa sequence that identical to an aa    sequence of a member of the pair of CH3 aa sequences selected from    the group consisting of SEQ ID NOs:31 and 35; SEQ ID NOs:33 and 37;    SEQ ID NOs:39 and 43; SEQ ID NOs:41 and 45; SEQ ID NOs:47 and 51;    SEQ ID NOs:49 and 53; SEQ ID NOs:55 and 59; SEQ ID NOs:57 and 61;    SEQ ID NOs:63 and 67; SEQ ID NOs:65 and 69; SEQ ID NOs:71 and 73;    SEQ ID NOs:72 and 74; SEQ ID NOs:75 and 79; SEQ ID NOs:77 and 81;    SEQ ID NOs:83 and 85; SEQ ID NOs:84 and 86; SEQ ID NOs:87 and 89;    SEQ ID NOs:88 and 90; SEQ ID NOs:91 and 93; SEQ ID NOs:92 and 94;    SEQ ID NOs:95 and 97; and SEQ ID NOs:96 and 98.-   90. The TFcBA of any one of embodiments 59-89, wherein the first    hinge comprises an aa sequence that differs in at most 3 aa    deletions, additions or substitutions from an aa sequence selected    from the group consisting of SEQ ID NOs:4, 18, 19, 20, 21, 22,    263-265 and 267-273.-   91. The TFcBA of embodiment 90, wherein the first hinge comprises an    aa sequence that is an aa sequence selected from the group    consisting of SEQ ID NOs:4, 18, 19, 20, 21, 22, 263-265 and 267-273-   92. The TFcBA of any one of embodiments 59-91, wherein the second    hinge comprises an aa sequence that differs in at most 3 aa    deletions, additions or substitutions from an aa sequence selected    from the group consisting of SEQ ID NOs:23, 24, 263-265 and 267-273.-   93. The TFcBA of embodiment 92, wherein the second hinge comprises    an aa sequence that is an aa sequence selected from the group    consisting of SEQ ID NOs:23, 24, 263-265 and 267-273.-   94. The TFcBA of any one of embodiments 59-93, comprising a CH2    domain comprising an aa sequence that is at least 70% identical to    SEQ ID NO:25, 26, 261 or 262, or which differs therefrom in at most    30 aa deletions, additions or substitutions.-   95. The TFcBA of any one of embodiments 1-94, wherein the TFc    comprises in amino to carboxyl terminal order: a first hinge, a    first CH2 domain, a first CH3 domain, a second hinge, a second CH2    domain and a second CH3 domain, wherein    -   a. the first hinge comprises an aa sequence selected from the        group consisting of SEQ ID NOs:4, 18, 19, 263-265 and 267-273;    -   b. the first CH2 domain is aglycosylated and comprises the aa        sequence set forth as SEQ ID NO:25;    -   c. the first CH3 domain comprises an aa sequence that is either        sequence of a pair of sequences selected from the group of pairs        of CH3 domain sequences consisting of SEQ ID NOs:31 and 35; SEQ        ID NOs:33 and 37; SEQ ID NOs:39 and 43; SEQ ID NOs:41 and 45;        SEQ ID NOs:47 and 51; SEQ ID NOs:49 and 53; SEQ ID NOs:55 and        59; SEQ ID NOs:57 and 61; SEQ ID NOs:63 and 67; SEQ ID NOs:65        and 69; SEQ ID NOs:71 and 73; SEQ ID NOs:72 and 74; SEQ ID        NOs:75 and 79; SEQ ID NOs:77 and 81; SEQ ID NOs:83 and 85; SEQ        ID NOs:84 and 86; SEQ ID NOs:87 and 89; SEQ ID NOs:88 and 90;        SEQ ID NOs:91 and 93; SEQ ID NOs:92 and 94; SEQ ID NOs:95 and        97; and SEQ ID NOs:96 and 98;    -   d. the second hinge comprises an aa sequence consisting of a        sequence selected from the group consisting of SEQ ID NO:23,        263-265 and 267-273;    -   e. the second CH2 domain is aglycosylated and comprises the aa        sequence set forth in SEQ ID NO:25; and    -   f. the second CH3 domain comprises an aa sequence that is either        sequence of a pair of sequences selected from the group of pairs        of CH3 domain sequences consisting of SEQ ID NOs:31 and 35; SEQ        ID NOs:33 and 37; SEQ ID NOs:39 and 43; SEQ ID NOs:41 and 45;        SEQ ID NOs:47 and 51; SEQ ID NOs:49 and 53; SEQ ID NOs:55 and        59; SEQ ID NOs:57 and 61; SEQ ID NOs:63 and 67; SEQ ID NOs:65        and 69; SEQ ID NOs:71 and 73; SEQ ID NOs:72 and 74; SEQ ID        NOs:75 and 79; SEQ ID NOs:77 and 81; SEQ ID NOs:83 and 85; SEQ        ID NOs:84 and 86; SEQ ID NOs:87 and 89; SEQ ID NOs:88 and 90;        SEQ ID NOs:91 and 93; SEQ ID NOs:92 and 94; SEQ ID NOs:95 and        97; and SEQ ID NOs:96 and 98, wherein if the first CH3 domain        comprises a first sequence of a pair of sequences, the second        CH3 domain comprises the second sequence of the pair of        sequences; and if the first CH3 domain comprises the second        sequence of a pair of sequences, the second CH3 domain comprises        the first sequence of the pair of sequences.-   96. The TFcBA of any one of embodiments 59-94, wherein the TFc    comprises in amino to carboxyl terminal order: a first hinge, a    first CH2 domain, a first CH3 domain, a second hinge, a second CH2    domain and a second CH3 domain, wherein    -   a. the first hinge comprises an aa sequence selected from the        group consisting of SEQ ID NOs:20, 21, 22, 263-265 and 267-273;    -   b. the first CH2 domain is aglycosylated and comprises the aa        sequence set forth in SEQ ID NO:26;    -   c. the first CH3 domain comprises an aa sequence that is either        sequence of a pair of sequences selected from the group of pairs        of CH3 domain sequences consisting of SEQ ID NOs:31 and 35; SEQ        ID NOs:33 and 37; SEQ ID NOs:39 and 43; SEQ ID NOs:41 and 45;        SEQ ID NOs:47 and 51; SEQ ID NOs:49 and 53; SEQ ID NOs:55 and        59; SEQ ID NOs:57 and 61; SEQ ID NOs:63 and 67; SEQ ID NOs:65        and 69; SEQ ID NOs:71 and 73; SEQ ID NOs:72 and 74; SEQ ID        NOs:75 and 79; SEQ ID NOs:77 and 81; SEQ ID NOs:83 and 85; SEQ        ID NOs:84 and 86; SEQ ID NOs:87 and 89; SEQ ID NOs:88 and 90;        SEQ ID NOs:91 and 93; SEQ ID NOs:92 and 94; SEQ ID NOs:95 and        97; and SEQ ID NOs:96 and 98;    -   d. the second hinge comprises an aa sequence consisting of SEQ        ID NO:24, 263-265 and 267-273;    -   e. the second CH2 domain is aglycosylated and comprises the aa        sequence set forth in SEQ ID NO:26; and    -   f. the second CH3 domain comprises an aa sequence that is either        sequence of a pair of sequences selected from the group of pairs        of CH3 domain sequences consisting of SEQ ID NOs:31 and 35; SEQ        ID NOs:33 and 37; SEQ ID NOs:39 and 43; SEQ ID NOs:41 and 45;        SEQ ID NOs:47 and 51; SEQ ID NOs:49 and 53; SEQ ID NOs:55 and        59; SEQ ID NOs:57 and 61; SEQ ID NOs:63 and 67; SEQ ID NOs:65        and 69; SEQ ID NOs:71 and 73; SEQ ID NOs:72 and 74; SEQ ID        NOs:75 and 79; SEQ ID NOs:77 and 81; SEQ ID NOs:83 and 85; SEQ        ID NOs:84 and 86; SEQ ID NOs:87 and 89; SEQ ID NOs:88 and 90;        SEQ ID NOs:91 and 93; SEQ ID NOs:92 and 94; SEQ ID NOs:95 and        97; and SEQ ID NOs:96 and 98, wherein if the first CH3 domain        comprises a first sequence of a pair of sequences, the second        CH3 domain comprises the second sequence of the pair of        sequences; and if the first CH3 domain comprises the second        sequence of a pair of sequences, the second CH3 domain comprises        the first sequence of the pair of sequences.-   97. The TFcBA of any one of embodiments 59-96, wherein the first or    the second Fc region comprises an aa sequence that is at least 70%    identical to an aa sequence selected from the group consisting of    SEQ ID NOs:99-166, or differs therefrom in at most 50 aa deletions,    additions or substitutions.-   98. The TFcBA of any one of embodiment 97, wherein the first or the    second Fc region comprises an aa sequence selected from the group    consisting of SEQ ID NOs:99-166.-   99. The TFcBA of embodiment 97, wherein either or both of the first    and the second Fc region comprises an aa sequence that is at least    70% identical to one aa sequence of a pair of aa sequences selected    from the group consisting of SEQ ID NOs:99 and 100; SEQ ID NOs:101    and 102; SEQ ID NOs:103 and 104; SEQ ID NOs:105 and 106; SEQ ID    NOs:107 and 108; SEQ ID NOs:109 and 110; SEQ ID NOs:111 and 112; SEQ    ID NOs:113 and 114; SEQ ID NOs:115 and 116; SEQ ID NOs:117 and 118;    SEQ ID NOs:119 and 120; SEQ ID NOs:121 and 122; SEQ ID NOs:123 and    124; SEQ ID NOs:125 and 126; SEQ ID NOs:127 and 128; SEQ ID NOs:129    and 130; SEQ ID NOs:131 and 132; SEQ ID NOs:133 and 134; SEQ ID    NOs:135 and 136; SEQ ID NOs:137 and 138; SEQ ID NOs:139 and 140; SEQ    ID NOs:141 and 142; SEQ ID NOs:143 and 144; SEQ ID NOs:145 and 146;    SEQ ID NOs:147 and 148; SEQ ID NOs:149 and 150; SEQ ID NOs:151 and    152; SEQ ID NOs:153 and 154; SEQ ID NOs:155 and 156; SEQ ID NOs:157    and 158; SEQ ID NOs:159 and 160; SEQ ID NOs:161 and 162; SEQ ID    NOs:163 and 164; and SEQ ID NOs:165 and 166, or which differs    therefrom in at most 50 aa deletions, additions or substitutions,    and wherein the first Fc region comprises a different member of the    pair than is comprised by the second Fc region.-   100. The TFcBA of embodiment 99, wherein the first Fc region and the    second Fc region together comprise a pair of two different members,    each member being an Fc aa sequence, wherein each pair is selected    from the group of pairs consisting of SEQ ID NOs:99 and 100; SEQ ID    NOs:101 and 102; SEQ ID NOs:103 and 104; SEQ ID NOs:105 and 106; SEQ    ID NOs:107 and 108; SEQ ID NOs:109 and 110; SEQ ID NOs:111 and 112;    SEQ ID NOs:113 and 114; SEQ ID NOs:115 and 116; SEQ ID NOs:117 and    118; SEQ ID NOs:119 and 120; SEQ ID NOs:121 and 122; SEQ ID NOs:123    and 124; SEQ ID NOs:125 and 126; SEQ ID NOs:127 and 128; SEQ ID    NOs:129 and 130; SEQ ID NOs:131 and 132; SEQ ID NOs:133 and 134; SEQ    ID NOs:135 and 136; SEQ ID NOs:137 and 138; SEQ ID NOs:139 and 140;    SEQ ID NOs:141 and 142; SEQ ID NOs:143 and 144; SEQ ID NOs:145 and    146; SEQ ID NOs:147 and 148; SEQ ID NOs:149 and 150; SEQ ID NOs:151    and 152; SEQ ID NOs:153 and 154; SEQ ID NOs:155 and 156; SEQ ID    NOs:157 and 158; SEQ ID NOs:159 and 160; SEQ ID NOs:161 and 162; SEQ    ID NOs:163 and 164; and SEQ ID NOs:165 and 166, each member aa    sequence being at least 70% identical to, or differing in at most 30    aa additions, deletions or substitutions from each sequence of each    said pair, wherein the first Fc region comprises a different member    of the pair than is comprised by the second Fc region.-   101. The TFcBA of any one of embodiments 59-100, comprising a TFc    comprising an aa sequence that is at least 70% identical to an aa    sequence selected from the group consisting of SEQ ID NOs:171, 173,    175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199,    201, 203, 205, 207, 209, 211, 213, 215, 217, 219 and 221 or which    differs therefrom in at most 30 aa additions, deletions or    substitutions.-   102. The TFcBA of embodiment 101, comprising a TFc comprising an aa    sequence selected from the group consisting of SEQ ID NOs:171, 173,    175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199,    201, 203, 205, 207, 209, 211, 213, 215, 217, 219 and 221.-   103. The TFcBA of any one of embodiments 59-102, comprising a heavy    chain that comprises in amino to carboxyl terminal order: a first    heavy chain variable (VH) domain, a TFc, a connecting linker and a    second VH domain-   104. The TFcBA of embodiment 103, wherein the heavy chain comprises    in amino to carboxyl terminal order: a first VH domain, a CH1    domain, a TFc, a connecting linker and a second VH domain-   105. The TFcBA of embodiment 104, wherein the heavy chain comprises    in amino to carboxyl terminal order: a first VH domain, a CH1    domain, a TFc, a connecting linker, a second VH domain, an scFv    linker and a second light chain variable (VL) domain, wherein the    second VH and VL domains associate to form a second binding site.-   106. The TFcBA of embodiment 105, comprising a light chain that    comprises a first VL domain that dimerizes with the first VH domain    to form a first binding site.-   107. The TFcBA of embodiment 106, wherein the light chain comprises    a light chain constant (CL) domain that is linked to the carboxyl    terminus of the VL domain-   108. The TFcBA of any one of embodiments 59-107, wherein the first    binding site is an anti-c-Met binding site and the second binding    site is an anti-EGFR binding site.-   109. A monovalent TFcA, comprising a binding site that is linked to    a TFc comprising a first Fc region and a second Fc region linked    through a TFc linker, wherein the first and the second Fc region    associate to form an Fc, and wherein either or both of the first and    the second Fc region comprise one or more aa modification to enhance    or stabilize the binding between the first and the second Fc region.-   110. A TFcA or TFcBA of any one of embodiments 1-109 that is a    charge-complementary paired TFcA or TFcBA, wherein    -   a charge-complementary paired TFcA or TFcBA is a TFcA or TFcBA        that comprises a pair of charged amino acids comprising an amino        acid selected from group A and an amino acid selected from group        B (a charge-complementary pair)    -   wherein group A comprises all natural amino acids with a pI of        greater than 7 and group B comprises all natural amino acids        with a pI of less than 7, or optionally wherein group A        comprises His, Lys, and Arg, and group B comprises Asp, Glu,        Asn, Phe, Gln, Tyr, Ser, Met, Thr, Ile, Gly, Val, Trp, Leu, Ala,        and Pro; and    -   said charge-complementary pair consists of a first amino acid        residue and a second amino acid residue, and    -   said charge-complementary pair is a position 297        charge-complementary pair or a position 299 charge-complementary        pair, wherein-   a position 297 charge-complementary pair is a charge-complementary    pair with said first amino acid residue located at EU position 297    of said first Fc region and said second amino acid residue located    at EU position 297 of said second Fc region, and a position 299    charge-complementary pair is a charge-complementary pair with said    first amino acid residue located at EU position 299 of said first Fc    region and said second amino acid residue located at EU position 299    of said second Fc region.-   111. The charge-complementary paired TFcA or TFcBA of embodiment    110, wherein the charge-complementary paired TFcA or TFcBA comprises    both a position 297 charge-complementary pair and a position 299    charge-complementary pair, wherein the first and second amino acid    residues of the position 297 charge-complementary pair are the same    as or different from the first and second amino acid residues of the    position 299 charge-complementary pair.-   112. The charge-complementary paired TFcA or TFcBA of embodiment 110    or 111, wherein the charge-complementary paired TFcA or TFcBA    comprises a position 297 charge-complementary pair and wherein the    charge-complementary paired TFcA or TFcBA is more stable than a TFcA    or TFcBA that is not a charge-complementary paired TFcA or TFcBA but    that is identical to the charge-complementary paired TFcA or TFcBA    except that amino acid residues corresponding to the first and the    second amino acid residues are both residues consisting of the same    charged amino acid, said same charged amino acid being one of the    amino acids of the position 297 charge-complementary pair of the    charge-complementary paired TFcA or TFcBA.-   113. The charge-complementary paired TFcA or TFcBA of embodiment    110, 111 or 112, wherein the charge-complementary paired TFcA or    TFcBA comprises a position 299 charge-complementary pair and wherein    the charge-complementary paired TFcA or TFcBA is more stable than a    TFcA or TFcBA that is not a charge-complementary paired TFcA or    TFcBA but that is identical to the charge-complementary paired TFcA    or TFcBA except that amino acid residues corresponding to the first    and the second amino acid residues are both residues consisting of    the same charged amino acid, said same charged amino acid being one    of the amino acids of the position 299 charge-complementary pair of    the charge-complementary paired TFcA or TFcBA.-   114. The TFcA or TFcBA of any one of embodiments 59-113, wherein the    first or the second binding site binds specifically to a human    protein selected from the group consisting of ErbB2, ErbB3, ErbB4,    IGF1R, IGF2R, Insulin receptor, Ron, c-Met, EGFR, VEGFR1, VEGFR2,    TNFR, FGFR1 FGFR2, FGFR3, FGFR4, PDGFR alpha, PDGFR beta, c-Kit,    EPCAM and EphA2.-   115. A pharmaceutical composition comprising a TFcA or TFcBA of any    one of embodiments 1-114 and a pharmaceutically acceptable carrier.-   116. A nucleic acid molecule comprising at least one coding    sequence, said at least one coding sequence encoding a heavy chain    or a light chain of a TFcA or TFcBA of any one of embodiments 1-114.-   117. A nucleic acid molecule comprising at least two coding    sequences, wherein one coding sequence encodes a heavy chain of a    TFcA or TFcBA of any one of embodiments 1-114 and a second coding    sequence encodes a light chain of the TFcBA.-   118. A vector comprising one or more nucleic acid molecules of    embodiments 116 or 117.-   119. A cell comprising one or more vectors of embodiment 118 or    nucleic acid molecule of embodiments 116 or 117.-   120. A cell comprising a nucleic acid molecule encoding the heavy    chain of a TFcA or TFcBA of any one of embodiments 1-114 and a    nucleic acid molecule encoding the light chain of the TFcA or TFcBA.-   121. A method of producing a TFcA or TFcBA comprising culturing the    host cell of embodiment 119 or 120 under conditions in which the    nucleic acids are expressed, and isolating the TFcA or TFcBA.-   122. A method for producing a TFcA or TFcBA, comprising culturing a    cell of embodiment 119 or 120 under conditions suitable for the    expression of the TFcA or TFcBA.-   123. A method of treating a subject having cancer, said method    comprising administering to a subject a therapeutically effective    amount of a TFcA or TFcBA, nucleic acid molecule, or vector of any    one of embodiments 1-120.    Further Exemplary Embodiments Include but are not Limited to the    Following Items in Connection with the Subject Matter which May be    Embodied.

A1′. A Tandem Fc Bispecific Antibody (“TFcBA”) that comprises twopolypeptide chains, a large chain and a Fab light chain, each chainhaving a C-terminus and an N-terminus, the TFcBA comprising a firstbinding site comprised by a Fab moiety comprising the Fab light chainand a Fab heavy chain, which Fab heavy chain is at the N-terminus of thelarge chain, which Fab moiety specifically binds to cMET, and whichTFcBA further comprises a second binding site comprised by a singlechain Fv (scFV) moiety at the C-terminus of the large chain, which scFvmoiety specifically binds to EpCAM, wherein:

(a) the Fab heavy chain and the scFv moiety are linked through a TandemFc (“TFc”);(b) the TFc is comprised by the large chain and has a first Fc regionand a second Fc region which are linked through a TFc linker to form acontiguous polypeptide; and(c) the first and the second Fc regions associate to form an Fc dimer,and further wherein(d) the Fab light chain comprises an amino acid sequence comprising thethree light chain CDRs present SEQ ID NO:400; and(e) the Fab heavy chain comprises an amino acid sequence comprising thethree heavy chain CDRs present in SEQ ID NO:423.

A2′. The TFcBA of embodiment A1′, wherein the scFV moiety comprises anamino acid sequence that is SEQ ID NO:488.

A3′. The TFcBA of embodiment A1′ or of embodiment A2′ wherein the TFcBAlarge chain comprises, in N-terminal to C-terminal order:

(i) the Fab heavy chain variable region IgG1 CH1 domain;

(ii) a hybrid hinge containing an IgG1 upper hinge and an IgG4 middleand lower hinge;

(iii) a first IgG4 CH2 domain comprising a T299K mutation;

(iv) an first IgG1 CH3 domain comprising T366S, L368A, and Y407Vmutations;

(v) a first disulfide bridge motif (KSCDKT);

(vi) a Tandem Fc linker, optionally a linker comprising the aa sequence(G₄5)₈;

(vii) an IgG4 middle and lower hinge;

(viii) a second IgG4 CH2 domain comprising a T299D mutation;

(ix) a second IgG1 CH3 domain comprising a T366W mutation;

(x) a second disulfide bridge motif (GEC);

(xi) a connecting linker; and

(xii) the scFv.

A4′. The TFcBA of any of embodiments A1′-A3′ wherein the TFcBA exhibitsa Kd for binding to cMET of less than about 10e-8, 10e-9, 10e-10,10e-11, 10e-12, or 10e-13 M.

A5′. The TFcBA of embodiment A4′ wherein the TFcBA exhibits a Kd forbinding to cMET of from 3.0×10e-8 to 2.5×10e-9 M.

A6′. The TFcBA of any of embodiments A1′-A3′ wherein the TFcBA exhibitsa Kd for binding to EpCAM of less than about 10e-7 or 10e-8, oroptionally 10e-9 M.

A7′. The TFcBA of embodiment A6′ wherein the TFcBA exhibits a Kd forbinding to cMET of from 10e-8 to 10e-11 M.

A8′. The TFcBA of any of embodiments A1′-A3′ wherein a TFc linkersequence comprises an amino acid sequence of SEQ ID NO:169.

A9′. The TFcBA of any of embodiments A1′-A3′ wherein the TFc comprisesan amino acid sequence selected from the group consisting of SEQ IDNO:171, SEQ ID NO:173, SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:179, SEQID NO:181, SEQ ID NO:183, SEQ ID NO:185, SEQ ID NO:187, SEQ ID NO:189,SEQ ID NO:191, SEQ ID NO:193 and SEQ ID NO:195.

A10′. A TFcBA molecule comprising a large chain comprising an amino acidsequence that is SEQ ID NO 489.

A11′. A method of treating a cancer patient, the method comprisingadministering to the patient a therapeutically effective amount of theTFcBA of any one of the preceding embodiments to the patient andoptionally wherein the therapeutically effective amount is from 100ng/kg of patient body weight to 15 mg/kg of patient body weight, andfurther optionally wherein the therapeutically effective amount is from1 mg/kg patient body weight to 25 mg/kg patient body weight.

A12′. The method of embodiment A11′, wherein the cancer is a type ofcancer disclosed herein and optionally wherein the therapeuticallyeffective amount is from 1 mg/kg of patient body weight to 10 mg/kg ofpatient body weight.

A13′. A method of inhibiting tumor growth in a cancer patient, themethod comprising administering to the patient a therapeuticallyeffective amount of a TFcBA of any one of the embodiments A1′ to A10′ tothe patient, optionally wherein the therapeutically effective amount isfrom 100 ng/kg of patient body weight to 15 mg/kg of patient bodyweight, from 1 mg/kg of patient body weight to 10 mg/kg of patient bodyweight, or from 1 mg/kg of patient body weight to 25 mg/kg of patientbody weight.

A14′. A method of inhibiting tumor cell proliferation, the methodcomprising contacting a tumor cell with a fluid comprising aconcentration of a TFcBA of any one of the embodiments A1′ to A10′,which concentration of the TFcBA is effective to inhibit proliferationof the tumor cells.

A15′. A pharmaceutical formulation comprising the TFcBA of any one ofembodiments A1′ to A10′ and a pharmaceutical carrier.

A16′. The pharmaceutical formulation of embodiment A15′, wherein theformulation is a sterile formulation suitable for injection, intravenousinjection and/or infusion.

A17′. The pharmaceutical formulation of embodiment A15′ or embodimentA16′, wherein the formulation is packaged in a pharmaceuticallyacceptable container.

A18′. A nucleic acid molecule encoding a protein amino acid sequence ofany of embodiments A1′ to A10′.

A19′. A vector comprising the nucleic acid molecule of embodiment A18′.

A20′. A host cell comprising a vector comprising the nucleic acidmolecule of embodiment A18′.

A21′. The host cell of embodiment A20′ wherein the host cell expressesthe encoded protein.

A22′. The TFcBA of any of embodiments A1-10′ wherein, when the TFcBA iscontacted with cells expressing both cell surface c-Met and cell surfaceEPCAM, the amount of total c-Met measured in the cells subsequent tocontact is lower than the amount of total c-Met measured in matchedcells prior to contact.

A23′. The TFcBA of embodiment A22′, wherein, when the TFcBA is contactedwith cells expressing both cell surface c-Met and cell surface EPCAM,the amount of total EPCAM measured in the cells is essentiallyunchanged.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Diagram of an exemplary anti-c-Met/anti-EGFR Tandem FcBispecific Antibody (“TFcBA”) (FIG. 1A) and exemplary mutations in eachof the domains of the Tandem Fc (“TFc”) (FIG. 1B).

FIG. 1A: Diagram of an exemplary anti-c-Met/anti-EGFR TFcBA comprisingthe following three modules in amino to carboxyl terminal order: 1) afirst module consisting of an anti-c-Met binding site; 2) a secondmodule consisting of a TFc; and 3) a third module consisting of ananti-EGFR binding site. In the exemplified TFcBA, the first module is ananti-c-Met Fab and the third module is an anti-EGFR scFv. The TFccomprises two Fc regions that are linked through a TFc linker. In theexemplified TFcBA, the first Fc region comprises a full length IgG1/IgG4hybrid hinge, an IgG4 CH2 domain, and an IgG1 CH3 domain, and the secondFc region comprises the core and lower hinge of IgG4 (but does notcomprise an upper hinge), an IgG4 CH2 domain and an IgG1 CH3 domain. Inthe exemplified TFcBA, the CH3 domain comprises one or more AssociationEnhancing Modifications (“AEMs”), which enhance the association betweentwo CH3 domains or two Fc regions. TFcBAs may also comprise one or moredisulfide bond forming modifications (“DiSs”), which introduce cysteinesallowing for the formation of disulfide bonds between two Fc regions.

FIG. 1B: Diagram of the structure of a TFc showing in amino to carboxylterminal order: the first hinge, the first CH2 domain, the first CH3domain, the TFc linker, the second hinge, the second CH2 domain and thesecond CH3 domain. Exemplary sequences and domain modifications for eachof these domains are shown below the diagram. The name of the first orsecond CH3 modification in each AEM or DiS is indicated in parenthesisafter the name of the modification, wherein the first numeral after“AEM” or “DiS” refers to the module number of the AEM or DiS,respectively, and the second numeral refers to the first or the secondof the two CH3 domains. For example, “AEM 1.1” is indicated after thesubstitutions “T366S/L368A/Y407V,” which substitutions are thecombination of substitutions in one of the two CH3 domains of a pair ofmodifications in AEM module 1. A TFc may comprise any combination ofeach of these domains, with the proviso that when one of the CH3 domainof the TFc comprises one of the two modifications of an AEM and/or DiS,the other CH3 domain comprises the second, i.e., compatible,modification(s) of the AEM and/or DiS. For example, if one CH3 domain ofa TFc comprises AEM 1.1, the other CH3 domain comprises AEM 1.2.“C-term. Cys” refers to a modification adding a C-terminal Cysteine tothe CH3 domain by substituting the last three aas of the CH3 domain withthose shown in the Figure. Aa residue numbers in this Figure and theother Figures are those in an intact antibody heavy chain, according tothe EU index in Kabat.

FIG. 2: Alignment of aa sequences of wild type and variant hinges. Adash “-” at a position represents an aa that is identical to that in thefirst line of the figure at that position. A) Aa sequences of fulllength (SEQ ID NOs:4, 18 and 19) or partial (SEQ ID NOs:1, 2, 3, 16, 17,23 and 263-265) IgG1 hinges that are wild type (SEQ ID NOs:1-4 and 23)or modified (SEQ ID NOs:16-19 and 263-265). B) Aas of full length (SEQID NOs:20, 21 and 22) or partial (SEQ ID NOs:1, 13, 14 and 24) IgG1/IgG4hybrid hinges that are wild type (SEQ ID NOs:1, 13, 14, 20 and 24) ormodified (SEQ ID NOs:21 and 22). C) Aa sequences of full length wildtype mIgG1 hinge (SEQ ID NO:266) and hybrid mIgG1/mIgG2A hinge (SEQ IDNO:267). D) Aa sequence of a full length, wild type hIgG2 hinge (SEQ IDNO:7) and modified hIgG2 hinges (SEQ ID NOs:268 and 269). E) Aa sequenceof a full length wild type hIgA2 hinge (SEQ ID NO:270) and modifiedhIgA2 hinges (SEQ ID NOs:271-273).

FIG. 3: Alignment of IgG1 CH3 aa sequences with or without various aamodifications. Each line is the aa sequence of a different CH3 domain. Adash “-” at a position represents an aa that is identical to that in thefirst line of the figure at that position. The CH3 modifications areorganized according to their module, e.g., AEM module 1. Each module isdivided into two groups labeled with two numerals: for example, AEMmodule 1 is divided into the groups “AEM 11” and “AEM 12,” wherein AEM11 represents the modifications made to one CH3 domain (domain “1”) ofmodule AEM 1 and AEM 12 represents the modifications made to the secondCH3 domain (domain “2”) of the module. Each line within a modulerepresents a CH3 domain having the modifications of the module with orwithout other modifications. The CH3 aa sequences within one modulediffer from each other, e.g., in the presence or absence of the carboxylterminal lysine and/or in the presence of the substitutions D356E andL358M.

FIG. 4: Alignment of exemplary IgG1 Fc regions. Each line is the aasequence of a different Fc region. A dash “-” at a position representsan aa that is identical to that in the first line of the figure at thatposition. Each Fc region comprises a hinge (boldface in first sequence),CH2 and CH3 domain (the CH3 domain is underlined in the first sequence).The SEQ ID NOs of the hinge, CH2 and CH3 sequences of each Fc in thisFigure are provided in Table 8. The Fcs are organized in pairs, whichare separated form other pairs by lines, and wherein each pairrepresents compatible Fcs, i.e., Fcs that can associate with each otherto form an Fc dimer.

FIG. 5: Alignment of exemplary IgG1/IgG4 hybrid Fc regions. Each line isthe aa sequence of a different Fc region. A dash “-” at a positionrepresents an aa that is identical to that in the first line of thefigure at that position. Each Fc region comprises a hinge (boldface inthe first sequence), CH2 and CH3 domain (the CH3 domain is underlined inthe first sequence). The SEQ ID NOs of the hinge, CH2 and CH3 sequencesof each Fc in this Figure are provided in Table 9. The Fcs are organizedin pairs, which are separated form other pairs by lines, and whereineach pair represents compatible Fcs, i.e., Fcs that can associate witheach other to form an Fc dimer.

FIG. 6: Aa sequences of the following IgG1 TFcs: 23 (SEQ ID NO:171); 23A(SEQ ID NO:173); 23B (SEQ ID NO:175); 23C (SEQ ID NO:177); 23D (SEQ IDNO:179); 23E (SEQ ID NO:181); 23F (SEQ ID NO:183); 23E (35L) (SEQ IDNO:185); 23E (35L Inverted) (SEQ ID NO:187); 23E (30L) (SEQ ID NO:189);23E (25L) (SEQ ID NO:191); 231 (SEQ ID NO:193); and 23J (SEQ ID NO:195).Each of these sequences consists of the following domains in amino tocarboxyl terminal order: a first IgG1 hinge (double underlined), an IgG1CH2 domain, an IgG1 CH3 domain (underlined), a (G4S)n linker (initalics), a second IgG1 hinge (doubly underlined, and consisting of thecore and lower hinges only), a second IgG1 CH2 domain and a second IgG1CH3 domain (underlined). The aa changes that are specific to each ofthese molecules are shown in boldface, and are named above the sequence.

FIG. 7: Aa sequences of the following IgG1/IgG4 hybrid TFcs: 39 (SEQ IDNO:197); 39A (SEQ ID NO:199); 39B (SEQ ID NO:201); 39C (SEQ ID NO:203);39D (SEQ ID NO:205); 39E (SEQ ID NO:207); 39F (SEQ ID NO:209); 39E (35L)(SEQ ID NO:211); 39E (35L Inverted) (SEQ ID NO:213); 39E (30L) (SEQ IDNO:215); 39E (25L) (SEQ ID NO:217); 39I (SEQ ID NO:219); 39J (SEQ IDNO:221). Each of these sequences consist of the following domains inamino to carboxyl terminal order: a first IgG1/IgG4 hybrid hingeconsisting of the IgG1 upper hinge and IgG4 core and lower hinges(double underlined), an IgG4 CH2 domain, an IgG1 CH3 domain(underlined), a (G4S)n linker (in italics), a second IgG4 hinge (doubleunderlined, and consisting of the core and lower hinges only), a secondIgG4 CH2 domain and a second IgG1 CH3 domain (underlined). The IgG1sequences are in upper case letters and the IgG4 sequences are in lowercase letters. The aa changes that are specific to each of thesemolecules are shown in boldface, and are named above the sequence.

FIG. 8: Samples of TFcs 23A, 23B, 23D, 23E, 39B and 39G separated on a4-12% SDS-PAGE gel under A) non reducing or B) reducing conditions.Molecular weights (in kilodaltons) of the molecular weight markers(Biorad Precision Plus Marker) of lane 1 are shown on the left of thegel.

FIG. 9: Aa sequences of heavy chains of the following exemplaryanti-c-Met/anti-EGFR TFcBAs: TFcBAs comprising a humanized 5D5 VH domainand an anti-EGFR scFv comprising the aa sequences of the VH and VLdomains of A), B), C), D) E), L) and M) panimumumab (SEQ ID NO:235); F)2224 (SEQ ID NO:239); G) cetuximab H1L1 (SEQ ID NO:260); H) cetuximabH1L2 (SEQ ID NO:281); I) cetuximab H2L1 (SEQ ID NO:283); and J)cetuximab H2L2 (SEQ ID NO:285). K) aa sequence of the heavy chain ofanti-c-Met/anti-EGFR TFcBA comprising the VH domain of anti-c-Metbinding site 2 and humanized anti-EGFR cetuximab scFv H1L1. The aas thatwere introduced into the cetuximab VH domains for humanization purposesare indicated in lower case. The CDRs of the anti-c-Met Fab areunderlined with a dotted line. The CH1 domain is underlined with a wavyline. The hinges are double underlined. The TFc linker is in italics.The CH3 domains are underlined. The AEM and DiS modifications in the CH3domains are in boldface. The scFv linker is in italics and underlined.The connecting linker is in italics and double underlined.

FIG. 10: Nucleotide sequences encoding the aa sequences set forth in theFigures and in the specification.

FIG. 11: Nucleotide and aa sequences of TFcs used in Examples 1 and 2.Each of the aa sequences consists of the following domains in amino tocarboxyl terminal order: a signal peptide (underlined and boldface), afirst IgG1 hinge (double underlined), an IgG1 CH2 domain, an IgG1 CH3domain (underlined), a TFc linker (in italics), a second IgG1 hinge(doubly underlined, and consisting of the core and lower hinges only), asecond IgG1 CH2 domain and a second IgG1 CH3 domain (underlined). IgG1aas are in upper case and IgG4 aas are in lower case. The aa changesthat are specific to each of these molecules, e.g., AEMs and DiSsmodifications, are shown in boldface, and are named above the sequence.

FIG. 12A: Selection of onartuzumab (OTZM) monoclonal cell line: Lane1=size standards, Lanes 2-12; 2=OTZM line 1, 3=OTZM line 2, 4=OTZM line3, 5=OTZM line 4, 6=OTZM line 5, 7=OTZM line 6, 8=OTZM line 7, 9=OTZMline 8, 10=OTZM line 9, 11=OTZM line 10, 12=OTZM line 11.

FIG. 12B: Selection of onartuzumab (OTZM) monoclonal cell line: Lane1=size standards Lanes 2-9; 2=OTZM line 12, 3=OTZM line 13, 4=OTZM line14, 5=OTZM line 15, 6=OTZM line 16, 7=OTZM line 17, 8=OTZM line 18,9=OTZM line 19.

FIG. 13A: Non-reduced SDS-PAGE of charged glycosylation mutants: Lane1=size standards, Lanes 2-8; 2=glyco wt, 3=glyco 1, 4=glyco 2, 5=glyco3, 6=glyco 4, 7=glyco 5, 8=glyco 6.

FIG. 13B: Reduced SDS-PAGE of charged glycosylation mutants: Lane 1=sizestandards, Lanes 2-8; 2=glyco wt, 3=glyco 1, 4=glyco 2, 5=glyco 3,6=glyco 4, 7=glyco 5, 8=glyco 6.

FIG. 14: Nucleotide and aa sequences of exemplary TFcBAs.

FIG. 15: A graph showing binding to cMet-Fc and EGFR-his of TFcBAscomprising the 39E glycoform 4 backbone, onartuzumab antibody and either2224 or panitumumab antibody.

FIG. 16: A graph showing inhibition of pMet by TFcs comprisingonartuzumab antibody and various backbones including 23, 23E, 39, 39Eglycoform 4 backbone, and including TFcBAs comprising 39E glycoform 4backbone and 2224, cetuximab, or panitumumab antibody.

FIG. 17: Nucleotide and aa sequences of glycosylation mutants of theexemplary TFcBAs set forth in Table 23.

FIG. 18: Diagram of an exemplary anti-c-Met/anti-EpCam Tandem FcBispecific Antibody (“TFcBA”). In an embodiment, the CH2 domain maycomprise an electrostatic region such as a complementarity region.

FIG. 19: Table showing structural characteristics ofanti-c-Met/anti-EpCam Tandem Fc Bispecific Antibodies Ab#1-Ab#13.

FIG. 20: SDS gel of variants of antibody OA-5D5 (IgGs).

FIG. 21: SDS gel of anti-c-Met/anti-EpCam Tandem Fc BispecificAntibodies Ab#5, Ab#7, and Ab#13.

FIG. 22: Bispecific binding assay data obtained withanti-c-Met/anti-EpCam Tandem Fc Bispecific Antibodies Ab#5, Ab#7, andAb#13.

FIG. 23: Comparison of Met phosphorylation induced by incubation withmonovalent (OA-5D5), bivalent anti-c-Met antibodies, HGF, or media onlycontrol.

FIG. 24: Comparison of Met phosphorylation induced by incubation of A549cells with monovalent (OA-5D5), bivalent anti-c-Met antibodies, HGF,anti-c-Met/anti-EpCam Tandem Fc Bispecific Antibodies Ab#5, Ab#7, orAb#13, or media only control.

FIG. 25A: Comparison of proliferation induced by incubation of A549cells with HGF, fetal bovine serum (FBS), and anti-c-Met/anti-EpCamTandem Fc Bispecific Antibodies Ab#5, Ab#7, or Ab#13.

FIG. 25B: Comparison of proliferation induced by incubation of H441cells with HGF, fetal bovine serum (FBS), and anti-c-Met/anti-EpCamTandem Fc Bispecific Antibodies Ab#5, Ab#7, or Ab#13.

FIG. 25C: Comparison of proliferation induced by incubation of HCC827cells with HGF, fetal bovine serum (FBS), and anti-c-Met/anti-EpCamTandem Fc Bispecific Antibodies Ab#5, Ab#7, or Ab#13.

FIG. 26: Comparison of inhibition of HGF-induced phosphorylation of AKTby monovalent anti-c-Met antibody OA-5D5, and anti-c-Met/anti-EpCamTandem Fc Bispecific Antibodies Ab#5, Ab#7, and Ab#13 in A549 cells andNCI-H2170 cells.

FIG. 27: Comparison of inhibition of HGF-induced proliferation bymonovalent anti-c-Met antibody OA-5D5, and anti-c-Met/anti-EpCam TandemFc Bispecific Antibody Ab#5 in U-87 MG cells and H441 cells.

FIG. 28: Comparison of inhibition of HGF-induced proliferation bymonovalent anti-c-Met antibody OA-5D5, and anti-c-Met/anti-EpCam TandemFc Bispecific Antibody Ab#7 in A549 cells and H441 cells.

FIG. 29: Downregulation of cMet levels in A549 cells and H2170 cells byanti-c-Met/anti-EpCam Tandem Fc Bispecific Antibody AB#5 but not bymonovalent anti-c-Met antibody OA-5D5.

FIG. 30A: Murine pharmacokinetics—terminal half life ofanti-c-Met/anti-EpCam Tandem Fc Bispecific Antibody Ab#5.

FIG. 30B: Murine pharmacokinetics—terminal half life ofanti-c-Met/anti-EpCam Tandem Fc Bispecific Antibody Ab#7.

FIG. 30C: Murine pharmacokinetics—terminal half life ofanti-c-Met/anti-EpCam Tandem Fc Bispecific Antibody Ab#13.

FIG. 31: illustrates the results of testing the effect of an agent ontumor volume over time. Anti-c-Met/anti-EpCam Tandem Fc BispecificAntibody Ab#7 and monovalent anti-c-Met antibody OA-5D5 each reduce U87MG cell xenograft tumor growth in nude mice and cause tumor regression.Both Ab#7 and OA-5D5 were able to demonstrate regression ofsubcutaneously implanted U-87 MG tumors to a similar extent.

FIG. 32 is a graph showing results of dose-response analysis of antibodyactivity on U-87 MG tumors implanted into nude mice, as measured bypost-implantation tumor volume. U-87 MG tumors were implantedsubcutaneously and the mice were treated weekly with PBS control or Ab#7at 1 mg/kg, 4 mg/kg, 12 mg/kg, or 24 mg/kg. The results show that AB#7produces a reduction in tumor volume with a dose response correlation:increasing doses of Ab#7 yield increasing degrees of reduction of tumorvolume.

FIG. 33 is a bar graph showing results of quantification of secreted HGFfrom tumor cells. HGF was measured in supernatants from U-87 MG cells,NCI-H358 cells (“parental”), NCI-H358 mock-transfected cells, andNCI-H358 cells transfected with HGF and normalized against totalprotein.

FIG. 34 is a graph showing results of dose-response analysis of antibodyactivity on HGF ligand-dependent HCC827-HGF tumors implanted into nudemice, as measured by post-implantation tumor volume. After tumor growth,mice were treated weekly with PBS control, Ab#7 at 1 mg/kg, 4 mg/kg, 12mg/kg (equimolar to 10 mg/kg OA-5D5), or 25 mg/kg, or 10 mg/kg OA-5D5.Results show that Ab#7 causes greater reductions in tumor volume thandoes and equimolar amount of OA-5D5.

FIG. 35 is a graph showing results of antibody activity on HGFligand-dependent H358-HGF tumors implanted into nude mice, as measuredby post-implantation tumor volume. After tumor growth, mice were treatedweekly with PBS control, Ab#7 at 12 mg/kg (equimolar to 10 mg/kgOA-5D5), or 10 mg/kg OA-5D5. Results show that Ab#7 causes greaterreductions in tumor volume than does and equimolar amount of OA-5D5.

FIG. 36 provides five graphs showing protein quantitation afterimmunoblot analysis of c-Met and EpCAM degradation in cell lysates aftertreatment with bispecific antibodies. Protein was measured in cellstreated with medium only, 200 nM OA-5D5, and 200 nM Ab#7. Part (A) showsquantification of c-Met in A549 cells; EpCAM was undetectable in thiscell line in this experiment. Part (B) shows quantitation of c-Met inNCI-H441 cells and (D) shows quantitation of EpCAM in NCI-H441 cells.Part (C) shows quantitation of c-Met in NCI-H2170 cells and (E) showsquantitation of EpCAM in NCI-H2170 cells. Results show that Ab#7, butnot OA-5D5, induces degradation of total c-Met in NCI-H441 and NCI-H2170cells, but not in A549 cells, while none of these antibodies inducedreductions in EPCAM levels in any of these cell lines.

BRIEF DESCRIPTION OF THE SEQUENCES

The amino acid (“aa”) sequences referred to herein and listed in thesequence listing are identified below.

SEQ ID NOs:1, 2 and 3 are the aa sequences of the wild type IgG1 upper,middle (or core) and lower hinge, respectively (see Table 2).

SEQ ID NO:4 is the aa sequence of the complete wild type IgG1 hinge,consisting of SEQ ID NOs:1, 2 and 3 in a contiguous sequence in amino tocarboxyl terminal order (see Table 2).

SEQ ID NOs:5 and 6 are the aa sequences of the wild type IgG2 upper andlower hinge, respectively (see Table 2). The IgG2 middle hinge is thesame as that of IgG1, i.e., SEQ ID NO:2.

SEQ ID NO:7 is the aa sequence of the complete wild type IgG2 hinge,consisting of SEQ ID NOs:5, 2 and 6 in a contiguous sequence in amino tocarboxyl terminal order (see Table 2).

SEQ ID NOs:8, 9 and 10 are the aa sequences of the wild type IgG3 upper,middle and lower hinge, respectively (see Table 2).

SEQ ID NO:11 is the aa sequence of the complete wild type IgG3 hinge,consisting of SEQ ID NOs:8, 9 and 10 in a contiguous sequence in aminoto carboxyl terminal order (see Table 2).

SEQ ID NOs:12, 13 and 14 are the aa sequences of the IgG4 upper, middleand lower hinge, respectively (see Table 2).

SEQ ID NO:15 is the aa sequence of a full length IgG4 hinge, consistingof SEQ ID NOs:12, 13 and 14 in a contiguous sequence in amino tocarboxyl terminal order (see Table 2).

SEQ ID NO:16 is the aa sequence of the IgG1 upper hinge (SEQ ID NO:1)comprising the aa substitutions H224C and T225C (see Table 4 and FIG.2).

SEQ ID NO:17 is the aa sequence of the IgG1 upper hinge (SEQ ID NO:1)comprising the aa substitution T223C (see Table 4 and FIG. 2).

SEQ ID NO:18 is the aa sequence of the full length IgG1 hinge (SEQ IDNO:4) comprising the aa substitutions H224C and T225C (see Table 4 andFIG. 2).

SEQ ID NO:19 is the aa sequence of the full length IgG1 hinge (SEQ IDNO:4) comprising the aa substitution T223C (see Table 4 and FIG. 2).

SEQ ID NO:20 is the aa sequence of a full length hybrid IgG1/IgG4 hinge,consisting of the upper hinge of IgG1 (SEQ ID NO:1) and the middle andlower hinges of IgG4 (SEQ ID NOs:13 and 14, respectively; see Table 4and FIG. 2).

SEQ ID NO:21 is the aa sequence of a full length hybrid IgG1/IgG4 hinge,consisting of the upper hinge of IgG1 comprising the aa substitutionsH224C and T225C (SEQ ID NO:16) and the middle and lower hinges of IgG4(SEQ ID NOs:13 and 14, respectively; see Table 4 and FIG. 2).

SEQ ID NO:22 is the aa sequence of a full length hybrid IgG1/IgG4 hinge,consisting of the upper hinge of IgG1 comprising the aa substitutionT223C (SEQ ID NO:17) and the middle and lower hinges of IgG4 (SEQ IDNOs:13 and 14, respectively; see Table 4 and FIG. 2).

SEQ ID NO:23 is the aa sequence of a partial IgG1 hinge comprising themiddle and lower IgG1 hinges (SEQ ID NOs:2 and 3), but not the upperhinge (see Table 4 and FIG. 2).

SEQ ID NO:24 is the aa sequence of a partial IgG4 hinge comprising themiddle and lower IgG4 hinges (SEQ ID NOs:13 and 14), but not the upperhinge (see Table 4 and FIG. 2).

SEQ ID NO:25 is the aa sequence of a full length IgG1 CH2 domain withthe aa substitution N297Q reducing glycosylation at aa 297.

SEQ ID NO:26 is the aa sequence of a full length wild type IgG4 CH2domain with the aa substitution T299K reducing glycosylation at aa 297.

SEQ ID NO:27 is the aa sequence of a full length wild type human IgG1CH3 domain (see Table 6 and FIG. 3).

SEQ ID NO:28 is the aa sequence of the wild type IgG1 CH3 domain havingSEQ ID NO:27, but lacking the C-terminal lysine (see Table 6 and FIG.3).

SEQ ID NO:29 is the aa sequence of the IgG1 CH3 domain having SEQ IDNO:27 with the substitutions D356E and L358M (see Table 6 and FIG. 3).

SEQ ID NO:30 is the aa sequence of the IgG1 CH3 domain having SEQ IDNO:29, lacking the C-terminal lysine (see Table 6 and FIG. 3).

SEQ ID NO:31 is the aa sequence of the IgG1 CH3 domain having SEQ IDNO:27 with the substitutions T366S, L368A and Y470V, creating a “hole”(Association Enhancing Modification or “AEM” 1.1; see Table 6 and FIG.3).

SEQ ID NO:32 is the aa sequence of the IgG1 CH3 domain having SEQ IDNO:31, lacking the C-terminal lysine (see Table 6 and FIG. 3).

SEQ ID NO:33 is the aa sequence of the IgG1 CH3 domain having SEQ IDNO:29 with the substitutions T366S, L368A and Y470V, creating a “hole”(AEM 1.1; see Table 6 and FIG. 3).

SEQ ID NO:34 is the aa sequence of the IgG1 CH3 domain having SEQ IDNO:33, lacking the C-terminal lysine (see Table 6 and FIG. 3).

SEQ ID NO:35 is the aa sequence of the IgG1 CH3 domain having SEQ IDNO:27 with the substitution T366W, creating a “bump” or “knob” (AEM 1.2;see Table 6 and FIG. 3).

SEQ ID NO:36 is the aa sequence of the IgG1 CH3 domain having SEQ IDNO:35, lacking the C-terminal lysine (see Table 6 and FIG. 3).

SEQ ID NO:37 is the aa sequence of the IgG1 CH3 domain having SEQ IDNO:29 with the substitution T366W, creating a “bump” or “knob” (AEM 1.2;see Table 6 and FIG. 3).

SEQ ID NO:38 is the aa sequence of the IgG1 CH3 domain having SEQ IDNO:37, lacking the C-terminal lysine (see Table 6 and FIG. 3).

SEQ ID NOs:39-98 are the aa sequences of IgG1 CH3 domains comprising oneor more AEM and/or Disulfide bond forming (“DiS”) modifications relativeto IgG1 CH3 having SEQ ID NO:27, 28, 29 or 30 (see Table 6 and FIG. 3).

SEQ ID NOs:99-132 are the aa sequences of exemplary IgG1 Fc regionscomprising in a contiguous amino to carboxyl terminal order: (a) a hingeselected from the group consisting of an IgG1 hinge, an IgG1 hingecomprising one or more aa substitutions, and a partial IgG1 hinge; (b)an IgG1 CH2 domain with N297Q (SEQ ID NO:25); and (c) an IgG1 CH3 domainselected from the group consisting of SEQ ID NO:29 and SEQ ID NO:29comprising one or more AEM and/or DiS modifications (FIG. 4). The hinge,CH2 and CH3 domains are covalently linked without intervening sequences.The SEQ ID NOs of each of the domains of SEQ ID NOs:99-132 are set forthin Table 8.

SEQ ID NOs:133-166 are the aa sequences of exemplary IgG1/IgG4 hybrid Fcregions comprising in a contiguous amino to carboxyl terminal order: (a)a hinge selected from the group consisting of an IgG1/IgG4 hybrid hinge,an IgG1/IgG4 hybrid hinge comprising one or more aa substitutions, and apartial IgG4 hinge; (b) an IgG4 CH2 domain with T299K (SEQ ID NO:26);and (c) an IgG1 CH3 domain selected from the group consisting of SEQ IDNO:29 and SEQ ID NO:29 comprising one or more AEM and/or DiSmodifications. The hinge, CH2 and CH3 domains are covalently linkedwithout intervening sequences. The SEQ ID NOs of each of the domains ofSEQ ID NOs:133-166 are set forth in Table 9.

SEQ ID NO:167 is KSCDKT, which is an exemplary modified carboxylterminal portion of an IgG1 CH3 domain that introduces a cysteine.

SEQ ID NO:168 is GEC, which is an exemplary modified carboxyl terminalportion of an IgG1 CH3 domain that introduces a cysteine.

SEQ ID NO:169 is the aa sequence of an exemplary non Gly-Ser TFc linker

SEQ ID NOs:170-195 are nucleotide sequences (even numbers) and aasequences (odd numbers) of exemplary IgG1 TFcs, which are set forth inFIG. 6. The SEQ ID NOs of the domains that constitute each of these IgG1TFcs is set forth in Table 12.

SEQ ID NOs:196-221 are nucleotide sequences (even numbers) and aasequences (odd numbers) of exemplary TFcs comprising hybrid IgG1/IgG4 Fcregions, which are set forth in FIG. 7. The SEQ ID NOs of the domainsthat constitute each of these hybrid TFcs is set forth in Table 13.

SEQ ID NOs:222-223 are the nucleotide and aa sequences, respectively, ofthe heavy chain Fab domain of anti-c-Met Ab 5D5, without signal peptide.

SEQ ID NOs:224-225 are the nucleotide and aa sequences, respectively, ofthe heavy chain of an IgG1 TFcBA comprising the anti-c-Met 5D5 VHdomain, an IgG1 TFc (with AEM 1), and the panitumumab scFv (FIG. 9).

SEQ ID NOs:226-227 are the nucleotide and aa sequences, respectively, ofthe heavy chain of an IgG1/IgG4 hybrid TFcBA comprising the anti-c-Met5D5 VH domain, an IgG1/IgG4 hybrid TFc (with AEM 1), and the panitumumabscFv (FIG. 9).

SEQ ID NOs:228-229 are the nucleotide and aa sequences, respectively, ofthe heavy chain of an IgG1/IgG4 hybrid TFcBA comprising the anti-c-Met5D5 VH domain, an IgG1/IgG4 hybrid TFc (with AEM 1), and the panitumumabscFv (FIG. 9).

SEQ ID NOs:230 and 231 are the nucleotide and aa sequences,respectively, of a light chain comprising humanized 5D5 anti-c-Met VLdomain and CL domain, for use, e.g., with a heavy chain comprising thehumanized 5D5 anti-c-Met VH domain, e.g., a heavy chain comprising SEQID NO: 225, 227, 229, 244, or 343.

SEQ ID NOs:232 and 233 are the nucleotide and aa sequences of ananti-EGFR scFv comprising the variable regions of panitumumab(VECTIBIX).

SEQ ID NOs:234 and 235 are the nucleotide and aa sequences,respectively, shown in FIGS. 9 and 10, respectively, of the heavy chainof an anti-c-Met/anti-EGFR TFcBA comprising (a) the anti-c-Met variabledomain from humanized 5D5; (b) a TFc with AEM 1 and DiS 2 (SEQ IDNO:181); and (c) an anti-EGFR scFv comprising the variable regions ofpanitumumab (VECTIBIX) (SEQ ID NO:233).

SEQ ID NOs:236 and 237 are the nucleotide and aa sequences,respectively, of an anti-EGFR scFv comprising the variable regions of Ab2224.

SEQ ID NOs:238 and 239 are the nucleotide and aa sequences, shown inFIGS. 9 and 10, respectively, of the heavy chain of ananti-c-Met/anti-EGFR TFcBA comprising (a) the anti-c-Met variable domainfrom humanized 5D5; (b) a TFc with AEM 1 and DiS 2 (SEQ ID NO:181); and(c) an anti-EGFR scFv comprising the variable regions of Ab 2224 (SEQ IDNO:237).

SEQ ID NOs:240 and 241 are the nucleotide and aa sequences,respectively, of an exemplary signal peptide.

SEQ ID NOs:242 and 243 are the nucleotide and aa sequence, respectively,of an exemplary signal peptide.

SEQ ID NOs:244 and 245 are the nucleotide and aa sequences,respectively, of the anti-c-Met VH domain of 5D5 and CL domain with asignal peptide having SEQ ID NO:241.

SEQ ID NO:246 and 247 are the nucleotide and aa sequences of the lightchain having SEQ ID NO:231 with a signal peptide having SEQ ID NO:243.

SEQ ID NOs:248-254 are the aa sequences of variant hinges described inthe specification.

SEQ ID NO: 255 and 256 are the nucleotide and aa sequences,respectively, of the heavy chain Fab region of the anti-c-Met bindingsite 2 (SEQ ID NO:287) with the signal peptide consisting of SEQ IDNO:241 and shown in Example 3.

SEQ ID NOs:257 and 258 are the nucleotide and aa sequences,respectively, of an anti-EGFR scFv comprising the variable regions ofhumanized cetuximab H1L1.

SEQ ID NOs:259 and 260 are the nucleotide and aa sequences, shown inFIGS. 9 and 10, respectively, of the heavy chain of ananti-c-Met/anti-EGFR TFcBA comprising (a) the anti-c-Met variable domainfrom humanized 5D5; (b) a TFc with AEM 1 and DiS 2 (SEQ ID NO:181); and(c) an anti-EGFR scFv comprising the variable regions of humanizedcetuximab H1L1 (SEQ ID NO:258).

SEQ ID NO:261 is the aa sequence of a full length wild type IgG1 CH2domain

SEQ ID NO:262 is the aa sequence of a full length wild type IgG4 CH2domain

SEQ ID NOs:263, 264 and 265 are aa sequences of variant hIgG1 hinges(FIG. 2).

SEQ ID NO:266 is the aa sequence of the wild type mouse IgG1 hinge (FIG.2).

SEQ ID NO:267 is the aa sequence of a mouse IgG1/IgG2A hybrid hinge(FIG. 2).

SEQ ID NOs:268 and 269 are the aa sequences of variant hIgG2 hinges(FIG. 2).

SEQ ID NO:270 is the aa sequence of a wild type hIgA2 hinge (FIG. 2).

SEQ ID NOs:271-273 are aa sequences of variant hIgA2 hinges (FIG. 2).

SEQ ID NOs:274-279 are nucleotide (even numbers) and aa (odd numbers)sequences of scFvs comprising variable domains of humanized cetuximabAbs H1L2, H2L1 and H2L2, which are described in Example 3.

SEQ ID NOs:280-285 are nucleotide (even numbers) and aa (odd numbers)sequences of the heavy chain of an anti-c-Met/anti-EGFR TFcBA comprising(a) the anti-c-Met variable domain from humanized 5D5; (b) an anti-EGFRscFv comprising the variable regions of humanized cetuximab Abs H1L2,H2L1 and H2L2 (SEQ ID NO:275, 277 or 279, respectively); and (c) a TFcwith AEM 1 and DiS 2 (SEQ ID NO:181) (FIG. 9).

SEQ ID NOs:286 and 287 are the nucleotide and aa sequences,respectively, of the heavy chain Fab domain of anti-c-Met binding site2, which is described in Example 3.

SEQ ID NOs:288 and 289 are the nucleotide and aa sequences,respectively, of the light chain Fab domain of anti-c-Met binding site2, which is described in Example 3.

SEQ ID NOs:290 and 291 are the nucleotide and aa sequences, shown inFIGS. 9 and 10, respectively, of the heavy chain of ananti-c-Met/anti-EGFR TFcBA comprising (a) the anti-c-Met heavy chain Fabdomain from anti-c-Met binding site 2 (SEQ ID NO:287); (b) a TFc withAEM 1 and DiS 2 (SEQ ID NO:181); and (c) an anti-EGFR scFv comprisingthe variable regions of humanized cetuximab H1L1 (SEQ ID NO:258) (FIG.9). The aa sequence of SEQ ID NO:291 is the same as that having SEQ IDNO:260, wherein the anti-c-Met binding domain has been replaced withthat of the anti-c-Met binding site 2.

SEQ ID NOs: 292-341 are nucleotide (even numbers) and aa (odd numbers)sequences of TFcs used in Examples 1 and 2 and shown in FIG. 11.

SEQ ID NOs: 342 and 343 are the nucleotide and aa sequences,respectively, of the heavy chain of an IgG1 TFcBA comprising theanti-c-Met 5D5 VH domain, an IgG1 TFc (with AEM 1 and DiS inverted), andthe panitumumab scFv (FIG. 9).

SEQ ID NO: 344 and 345 are the nucleotide and aa sequences,respectively, of the light chain Fab region of the anti-c-Met bindingsite 2 (SEQ ID NO:289) with the signal peptide consisting of SEQ IDNO:243 and shown in Example 3.

SEQ ID NOs: 346 and 347 are the nucleotide and aa sequences,respectively, of the heavy chain of anti-c-met/anti-EGFR TFcBA withhumanized 5D5 anti-c-Met and anti-EGFR panitumumab scFv with IgG1 TFc(with AEM 1 and a 40aa TFc linker having SEQ ID NO:169; FIG. 9).

SEQ ID NOs: 348 and 349 are the nucleotide and aa sequences,respectively, of the heavy chain of anti-c-met/anti-EGFR TFcBA withhumanized 5D5 anti-c-Met and anti-EGFR panitumumab scFv with IgG1/IgG4hybrid TFc (with AEM 1 and a 40aa TFc linker having SEQ ID NO:169; FIG.9).

SEQ ID NO: 350 is the aa sequence of the heavy chain ofanti-RON/anti-EGFR TFcBA comprising an anti-RON heavy chain Fab domain,anti-EGFR scFv 2224, and TFc 23E (SEQ ID NO:303); FIG. 14.

SEQ ID NO: 351 is the aa sequence of the heavy chain of theanti-RON/anti-EGFR TFcBA comprising an anti-RON heavy chain Fab domain,anti-EGFR scFv 2224, and TFc 39Egy4 (39E glycoform 4) (SEQ ID NO: 394);FIG. 14.

SEQ ID NO: 352 is the aa sequence of the heavy chain of theanti-RON/anti-CEA TFcBA comprising an anti-RON heavy chain Fab domain,anti-CEA scFv, and Tfc 23E (SEQ ID NO: 303); FIG. 14.

SEQ ID NO: 353 is the aa sequence of the heavy chain of theanti-RON/anti-CEA TFcBA comprising an anti-RON heavy chain Fab domain,anti-CEA scFv, and TFc 39Egy4 (SEQ ID NO: 394); FIG. 14.

SEQ ID NO: 354 is the aa sequence of the heavy chain of theanti-CEA/anti-cMet TFcBA comprising an anti-CEA heavy chain Fab domain,anti-cMet scFv, and TFc 23E (SEQ ID NO: 303); FIG. 14.

SEQ ID NO: 355 is the aa sequence of the heavy chain of theanti-CEA/anti-RON TFcBA comprising an anti-CEA heavy chain Fab domain,anti-RON scFv, and TFc 23E (SEQ ID NO: 303); FIG. 14.

SEQ ID NO: 356 is the aa sequence of the heavy chain of theanti-CEA/anti-scMet TFcBA comprising an anti-CEA heavy chain Fab domain,anti-cMet scFv, and TFc 39Egy4 (SEQ ID NO: 394); FIG. 14.

SEQ ID NOs 357-358 are the aa sequence and nucleotide sequence of TFcwild-type CH2 sequence; and T366S/L368A/Y407V/CH3 C-terminal CysteineKSCDKT::T366W/CH3 C-terminal Cysteine GEC in the CH3 domains; FIG. 17.

SEQ ID NO: 359 is the aa sequence of the heavy chain of theanti-cMet/anti-CEA TFcBA comprising an anti-cMet heavy chain Fab domain,anti-CEA scFv, and TFc 23E (SEQ ID NO: 303); FIG. 14.

SEQ ID NO: 360 is the aa sequence of the heavy chain of theanti-cMet/anti-CEA TFcBA comprising an anti-cMet heavy chain Fab domain,anti-CEA scFv, and TFc 39Egy4 (SEQ ID NO: 394); FIG. 14.

SEQ ID NO: 361 is the aa sequence of the heavy chain of theanti-cMet/anti-CEA CD44 comprising an anti-cMet heavy chain Fab, ananti-CD44 scFv, and TFc 39Egy4 (SEQ ID NO: 394); FIG. 14.

SEQ ID NO: 362 is the aa sequence of the heavy chain of theanti-cMet/anti-CEA CD44 comprising an anti-cMet heavy chain Fab domain,an anti-CD44 scFv, and TFc 23E (SEQ ID NO: 303); FIG. 14.

SEQ ID NO: 363 is the aa sequence of the heavy chain of theanti-cMet/anti-CEA CD44 comprising an anti-cMet heavy chain Fab domain,an anti-CD44 scFv, and TFc 23E (SEQ ID NO: 303); FIG. 14.

SEQ ID NO: 364 is the aa sequence of the heavy chain of theanti-cMet/anti-CEA CD44 comprising an anti-cMet heavy chain Fab domain,an anti-CD44 scFv, and TFc 39Egy4 (SEQ ID NO: 394), FIG. 14.

SEQ ID NO: 365 is the aa sequence of the heavy chain of theanti-CD44/anti-anti-cMet comprising an anti-CD44 heavy chain Fab domain,an anti-cMet scFv, and TFc 23E (SEQ ID NO: 303); FIG. 14.

SEQ ID NO: 366 is the aa sequence of the heavy chain of theanti-CD44/anti-cMet comprising an anti-CD44 heavy chain Fab domain, ananti-cMet scFv, and TFc 39Egy4 (SEQ ID NO: 394); FIG. 14.

SEQ ID NO: 367 is the aa sequence of the anti-CD44 ARH60-16-2 lightchain.

SEQ ID NOs 368-369 are the aa sequence and nucleotide sequence of theanti-cMet antibody onartuzumab and TFc 23 light chain; FIG. 14.

SEQ ID NOs 370-371 are the aa sequence and nucleotide sequence of theanti-cMet antibody onartuzumab and TFc 39 heavy chain; FIG. 14.

SEQ ID NOs 372-373 are the aa sequence and nucleotide sequence of theanti-cMet antibody onartuzumab and TFc 23E heavy chain; FIG. 14.

SEQ ID NOs 374-375 are the aa sequence and nucleotide sequence of theanti-cMet antibody onartuzumab and TFc 39Egy4 heavy chain; FIG. 14.

SEQ ID NOs 376-377 are the aa sequence and nucleotide sequence ofanti-cMet/anti-EGFR comprising an anti-cMet heavy chain Fab domain, thecetuximab anti-EGFR scFv, and TFc 23E (SEQ ID NO: 303); FIG. 14.

SEQ ID NOs 378-379 are the aa sequence and nucleotide sequence ofanti-cMet/anti-EGFR comprising an anti-cMet heavy chain Fab domain, thepanitumumab anti-EGFR scFv, and TFc 23E (SEQ ID NO: 303); FIG. 14.

SEQ ID NOs 380-381 are the aa sequence and nucleotide sequence ofanti-cMet/anti-EGFR comprising an anti-cMet heavy chain Fab domain, the2224 anti-EGFR scFv, and TFc 23E (SEQ ID NO: 303); FIG. 14.

SEQ ID NOs 382-383 are the aa sequence and nucleotide sequence ofanti-cMet/anti-EGFR comprising an anti-cMet heavy chain Fab domain thecetuximab anti-EGFR scFv, and TFc 39Egy4 (SEQ ID NO: 394); FIG. 14.

SEQ ID NOs 384-385 are the aa sequence and nucleotide sequence ofanti-cMet/anti-EGFR comprising an anti-cMet heavy chain Fab domain, thepanitumumab anti-EGFR scFv, and TFc 39Egy4 (SEQ ID NO: 394); FIG. 14.

SEQ ID NOs 386-387 are the aa sequence and nucleotide sequence ofanti-cMet/anti-EGFR comprising an anti-cMet heavy chain Fab domain, the2224 anti-EGFR scFv, and TFc 39Egy4 (SEQ ID NO: 394); FIG. 14.

For the sequences disclosed in FIG. 17, the double underline is thehinge, the single underline is the CH3 domain, the second doubleunderline is the second hinge, the second underline is the second CH3.

SEQ ID NOs 388-389 are the aa sequence and nucleotide sequence ofglycosylation mutant 1, comprising N297D/T299S::N297D/T299S amino acidchanges in the CH2 domains (underlined, bold-face), andT366S/L368A/Y407V/CH3 C-terminal Cysteine KSCDKT::T366W/CH3 C-terminalCysteine GEC in the CH3 domains; FIG. 17SEQ ID NOs 390-391 are the aasequence and nucleotide sequence of glycosylation mutant 2, comprisingT299K::N297D/T299S amino acid changes in the CH2 domains (underlined,bold-face), and T366S/L368A/Y407V/CH3 C-terminal CysteineKSCDKT::T366W/CH3 C-terminal Cysteine GEC in the CH3 domains; FIG. 17.

SEQ ID NOs 392-393 are the aa sequence and nucleotide sequence ofglycosylation mutant 3, comprising N297D/T299S::T299K amino acid changesin the CH2 domains (underlined, bold-face), and T366S/L368A/Y407V/CH3C-terminal Cysteine KSCDKT::T366W/CH3 C-terminal Cysteine GEC in the CH3domains; FIG. 17.

SEQ ID NOs 394-395 are the aa sequence and nucleotide sequence ofglycosylation mutant 4, comprising T299K::T299D amino acid changes inthe CH2 domains (underlined, bold-face), and T366S/L368A/Y407V/CH3C-terminal Cysteine KSCDKT::T366W/CH3 C-terminal Cysteine GEC in the CH3domains; FIG. 17.

SEQ ID NOs 396-397 are the aa sequence and nucleotide sequence ofglycosylation mutant 5, comprising T299D::T299K amino acid changes inthe CH2 domains (underlined, bold-face), and T366S/L368A/Y407V/CH3C-terminal Cysteine KSCDKT::T366W/CH3 C-terminal Cysteine GEC in the CH3domains; FIG. 17.

SEQ ID NOs 398-399 are the aa sequence and nucleotide sequence ofglycosylation mutant 6, comprising T299D::T299D amino acid changes inthe CH2 domains (underlined, bold-face), and T366S/L368A/Y407V/CH3C-terminal Cysteine KSCDKT::T366W/CH3 C-terminal Cysteine GEC in the CH3domains; FIG. 17.

SEQ ID NOs 400-489 are described below.

DETAILED DESCRIPTION

Provided herein are Tandem Fc Antibodies (“TFcAs”), e.g., Tandem FcBispecific Antibodies (“TFcBAs”). The molecules may be used for treatinga cell proliferative disorder, e.g., a cancer.

DEFINITIONS

For convenience, the meaning of certain terms and phrases used in thespecification, examples, and appended claims, are provided below.

“aa modification” or “aa change” refers to one or more amino acid (aa)deletion, addition or substitution to an aa sequence. Aa sequenceinsertions include amino- and/or carboxyl-terminal fusions ranging inlength from one residue to polypeptides containing a hundred or moreresidues, as well as intrasequence insertions of single or multiple aaresidues. Intrasequence insertions may range generally from about 1 to10 residues, e.g., 1 to 5, e.g., 1 to 3.

“AEM” or “association enhancing modification” refers to an aamodification made to a CH3 domain to enhance its association withanother CH3 domain. An AEM may comprise one or more aa substitutions,deletions or additions in one or both Fcs of a TFc. AEMs are classifiedin modules, e.g., module 1 (“AEM 1”), wherein the modification to one ofthe two CH3 domains is referred to as AEM 1.1 and the modification tothe other CH3 domain is referred to as AEM 1.2. For example, AEM 1.1consists of the combination of substitutions T366S/L368A and Y407V andAEM 1.2 consists of the aa substitution T366W. When a CH3 domaincomprises two or more aa modifications, e.g., aa substitutions, themodifications are separated from each other by a “/”. When referring tomodifications in two CH3 domains, the modifications in each of the CH3domains are separated by “::”.

An “anti-c-Met binding site” refers to a binding site that bindsspecifically to human c-Met.

An “anti-EGFR binding site” refers to a binding site that bindsspecifically to human EGFR.

“Antigen binding site” refers to a binding site that comprises the VHand/or VL domain of an antibody, or at least one CDR thereof, providedthat the antigen binding site binds specifically to its target antigen.For example, an antigen binding site may comprise, consist essentiallyof, or consist of a VHCDR3 alone or together with a VHCDR2 andoptionally a VHCDR1. In certain embodiments, an antigen binding sitecomprises a VH domain and a VL domain, which may be present on the samepolypeptide or on two different polypeptides, e.g., the VH domain ispresent on a heavy chain and a VL domain is present on a light chain.

“Antigen-binding portion” of an antibody refers to one or more fragmentsof an antibody that retain the ability to specifically bind to anantigen (e.g., c-met or EGFR). It has been shown that theantigen-binding function of an antibody can be retained by fragments ofa full-length antibody. Examples of binding fragments encompassed withinthe term “antigen-binding portion” of an antibody include (i) a Fabfragment, a monovalent fragment consisting of the VL, VH, CL and CH1domains; (ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) an Fdfragment consisting of the VH and CH1 domains; (iv) an Fv fragmentconsisting of the VL and VH domains of a single arm of an antibody, (v)a dAb fragment which consists of a VH domain; and (vi) an isolatedComplementarity Determining Region (“CDR”). Furthermore, although VL andVH are two domains of an Fv fragment, VL and VH are coded for byseparate genes, they can be joined, using recombinant methods, by asynthetic linker that enables them to be made as a single protein chainin which the VL and VH regions pair to form monovalent proteins, knownas single chain Fvs (scFvs) (see, e.g., U.S. Pat. No. 5,892,019). Suchsingle chain antibodies are also intended to be encompassed within theterm “antigen-binding portion” of an antibody. Other forms of singlechain antibodies, such as diabodies are also encompassed. Diabodies arebivalent, bispecific antibodies in which VH and VL domains are expressedon a single polypeptide chain, but using a linker that is too short toallow for pairing between the two domains on the same chain, therebyforcing the domains to pair with complementary domains of another chainand creating two antigen binding sites.

“Binding affinity” refers to the strength of a binding interaction andincludes both the actual binding affinity as well as the apparentbinding affinity. The actual binding affinity is a ratio of theassociation rate over the disassociation rate. The apparent affinity caninclude, for example, the avidity resulting from a polyvalentinteraction. Dissociation constant (Kd), is typically the reciprocal ofthe binding affinity, and may be conveniently measured using a surfaceplasmon resonance assay (e.g., as determined using a ForteBio Octetplatform (Pall ForteBio Corp.) or a BIACORE 3000 instrument (GEHealthcare) e.g., using recombinant c-Met as the analyte and ananti-c-Met antibody as the ligand) or a cell binding assay, examples ofof which assays is described in Example 3 of U.S. Pat. No. 7,846,440.

“Binding moiety,” “binding domain,” or “binding site,” refers to theportion, region, or site of a binding polypeptide or, when so specified,of a heavy or light chain thereof, that is directly involved inmediating the specific binding of an antibody to a target molecule(i.e., an antigen). Exemplary binding domains include an antigen bindingsite, a receptor binding domain of a ligand, a ligand binding domain ofa receptor or an enzymatic domain. In preferred embodiments, the bindingdomain comprises or consists of an antigen binding site (e.g.,comprising a variable heavy (VH) chain sequence and variable light (VL)chain sequence or six CDRs from an antibody placed into alternativeframework regions (e.g., human framework regions optionally comprisingone or more aa substitutions). In certain embodiments, a binding sitemay be comprised essentially only of a VH or a VL chain sequence. Abinding site may be entirely from one species, e.g., it has onlysequences that derive from the germline sequences of one species. Forexample, a binding site may be human (i.e., from the human species),mouse, or rat. A binding site may also be humanized, i.e., the CDRs arefrom one species and the frameworks (FRs) are from another species. Forexample, a binding site may have CDRs that were derived from a mouseantibody and FRs that are from the human species. Certain humanizedbinding sites comprise mutations in one or more CDR to make the CDRslook more like the CDRs of the donor antibody. Certain humanizedantibodies may also comprise mutations in one or more FR. Generallymutations in a binding site may enhance the affinity of binding of thebinding site to its target antigen, and/or they may stabilize thebinding site, e.g., to extend its half-life.

“CDR” or “complementarity determining region” refers to thenoncontiguous antigen combining sites found within the variable regionof both heavy and light chain polypeptides. These particular regionshave been described by Kabat et al., J. Biol. Chem. 252, 6609-6616(1977) and Kabat et al., Sequences of protein of immunological interest.(1991), and by Chothia et al., J. Mol. Biol. 196:901-917 (1987) and byMacCallum et al., J. Mol. Biol. 262:732-745 (1996) where the definitionsinclude overlapping or subsets of aa residues when compared against eachother. The aa residues which encompass the CDRs as defined by each ofthe above cited references are set forth for comparison. As used herein,and if not otherwise specified, “CDR” is as defined by Kabat. When CDRs(e.g., VH or VL CDRs) are described within larger sequences herein, theyare always arranged in N-terminal to C-terminal order as CDR1, CDR2,CDR3, and are separated by framework amino acid sequences.

TABLE 1 CDR definitions CDR Definitions Kabat¹ Chothia² MacCallum³VHCDR1 31-35 26-32 30-35 VHCDR2 50-65 53-55 47-58 VHCDR3  95-102  96-101 93-101 VLCDR1 24-34 26-32 30-36 VLCDR2 50-56 50-52 46-55 VLCDR3 89-9791-96 89-96 ¹Residue numbering follows the nomenclature of Kabat et al.,1991, supra ²Residue numbering follows the nomenclature of Chothia etal., supra ³Residue numbering follows the nomenclature of MacCallum etal., supra

“CH1 domain” refers to the heavy chain immunoglobulin constant domainlocated between the VH domain and the hinge. It spans EU positions118-215. A CH1 domain may be a naturally occurring CH1 domain, or anaturally occurring CH1 domain in which one or more amino acids (“aas”)have been substituted, added or deleted, provided that the CH1 domainhas the desired biological properties. A desired biological activity maybe a natural biological activity, an enhanced biological activity or areduced biological activity relative to the naturally occurringsequence.

“CH2 domain” refers to the heavy chain immunoglobulin constant domainthat is located between the hinge and the CH3 domain. As defined here,it spans EU positions 237-340. A CH2 domain may be a naturally occurringCH2 domain, or a naturally occurring CH2 domain in which one or more aashave been substituted, added or deleted, provided that the CH2 domainhas the desired biological properties. A desired biological activity maybe a natural biological activity, an enhanced biological activity or areduced biological activity relative to that of the naturally occurringdomain

“CH3 domain” refers to the heavy chain immunoglobulin constant domainthat is located C-terminally of the CH2 domain and spans approximately110 residues from the N-terminus of the CH2 domain, e.g., aboutpositions 341-446b (EU numbering system). A CH3 domain may be anaturally occurring CH3 domain, or a naturally occurring CH3 domain inwhich one or more aas have been substituted, added or deleted, providedthat the CH3 domain has the desired biological properties. A desiredbiological activity may be a natural biological activity, an enhancedbiological activity or a reduced biological activity relative to that ofthe naturally occurring domain A CH3 domain may or may not comprise aC-terminal lysine.

“CH4 domain” refers to the heavy chain immunoglobulin constant domainthat is located C-terminally of the CH3 domain in IgM and IgEantibodies. A CH4 domain may be a naturally occurring CH4 domain, or anaturally occurring CH4 domain in which one or more aas have beensubstituted, added or deleted, provided that the CH4 domain has thedesired biological properties. A desired biological activity may be anatural biological activity, an enhanced biological activity or areduced biological activity relative to that of the naturally occurringdomain

“CL domain” refers to the light chain immunoglobulin constant domainthat is located C-terminally to the VL domain. It spans about Kabatpositions 107A-216. A CL domain may be a naturally occurring CL domain,or a naturally occurring CL domain in which one or more aas have beensubstituted, added or deleted, provided that the CL domain has thedesired biological properties. A desired biological activity may be anatural biological activity, an enhanced biological activity or areduced biological activity relative to that of the naturally occurringdomain A CL domain may or may not comprise a C-terminal lysine.

“c-Met” or “c-MET” refers to Mesenchymal-Epithelial Transition (MET)factor, which is also known as Hepatocyte Growth Factor Receptor (HGFR),Scatter Factor (SF) receptor, AUTS9, RCCP2, corresponds to Gene ID 4233,and has tyrosine-kinase activity. The primary single chain precursorprotein is post-translationally cleaved to produce the alpha and betasubunits, which are disulfide linked to form the mature receptor. Twotranscript variants encoding different isoforms have been found for thisgene. HGF is the only known ligand for c-Met. The aa sequence of thehuman c-Met isoform a precursor is provided at Genbank Accession No.NP_001120972.1 and isoform b precursor is provided at Genbank AccessionNo. NP_000236.2.

A “constant region” or domain of a light chain of an immunoglobulin isreferred to interchangeably as a “CL,” “light chain constant regiondomain,” “CL region” or “CL domain.” A constant domain on a heavy chain(e.g. hinge, CH1, CH2 or CH3 domains) of an immunoglobulin is referredto interchangeably as a “CH,” “heavy chain constant domain,” “CH” regionor “CH domain” A variable domain on an immunoglobulin light chain isreferred to interchangeably as a “VL,” “light chain variable domain,”“VL region” or “VL domain” A variable domain on an immunoglobulin heavychain is referred to interchangeably as a “VH,” “heavy chain variabledomain,” “VH region” or “VH domain.”

“DiS” refers to the modification of a domain, e.g., a hinge or CH3domain, that results in the addition of a Cysteine, which can form adisulfide bond with another Cysteine. A DiS may comprise one or more aasubstitutions, deletions or additions in one or both Fcs of a TFc. DiSsare classified in modules, e.g., module 1 (“DiS 1”), wherein themodification to one of the two Fcs is referred to as DiS 1.1 and themodification to the other Fc is referred to as DiS 1.2. For example, DiS1.1 consists of the substitution Y349C and DiS 1.2 consists of the aasubstitution S354C.

“Domain” refers generally to a region, e.g., an independently folding,globular region or a non-globular region (e.g., a linker domain), of aheavy or light chain polypeptide which may comprise peptide loops (e.g.,1 to 4 peptide loops) that may be stabilized, for example, by aβ-pleated sheet and/or an intrachain disulfide bond. The constant andvariable regions of immunoglobulin heavy and light chains are typicallyfolded into domains. In particular, each one of the CH1, CH2, CH3, CH4,CL, VH and VL domains typically form a loop structure.

“EC₅₀” or “EC50” refers to the concentration of a molecule, e.g., aTFcA, that provides 50% of the maximal effect of the protein on aparticular system such as a binding assay or a signal transductionpathway.

“EGFR” refers to Epidermal Growth Factor Receptor, which is also knownas ErbB1, HER-1, mENA, and PIG61. EGFR is known to bind ligandsincluding epidermal growth factor (EGF), transforming growth factor α(TGf-α), amphiregulin, heparin-binding EGF (hb-EGF), betacellulin,epiregulin and has Gene ID 1956 (Herbst, R. S., and Shin, D. M., Cancer94 (2002) 1593-1611; Mendelsohn, J., and Baselga, J., Oncogene 19 (2000)6550-6565). EGFR is transmembrane glycoprotein that is a member of theprotein kinase superfamily that regulates numerous cellular processesvia tyrosine-kinase mediated signal transduction pathways, including,but not limited to, activation of signal transduction pathways thatcontrol cell proliferation, differentiation, cell survival, apoptosis,angiogenesis, mitogenesis, and metastasis (Atalay, G., et al., Ann.Oncology 14 (2003) 1346-1363; Tsao, A. S., and Herbst, R. S., Signal 4(2003) 4-9; Herbst, R. S., and Shin, D. M., Cancer 94 (2002) 1593-1611;Modjtahedi, H., et al., Br. J. Cancer 73 (1996) 228-235). Binding of theligand to EGFR induces receptor dimerization and tyrosineautophosphorylation, which leads to cell proliferation. Multiplealternatively spliced transcript variants that encode different proteinisoforms have been found for this gene. The aa sequences for human EGFRisoforms a-d precursors are provided at Genbank Accession Nos.NP_005219.2, NP_958439.1, NP_958440.1 and NP_958441.1.

“EpCAM” refers to epithelial cell adhesion molecule, a protein that inhumans is encoded by the EPCAM gene. EpCAM has also been designated asTACSTD1 (tumor-associated calcium signal transducer 1), CD326 (clusterof differentiation 326) and the 17-1A antigen. EpCAM is a pan-epithelialdifferentiation antigen that is expressed by most carcinomas.

“ErbB2” or “HER2” refers to a putative tyrosine kinase growth factorreceptor EGFR2, p185 HER2/NEU antigen, similar to the EGF receptor. Theaa sequences for ErbB2 isoforms are provided at Genbank Accession Nos.NP_004439.2 and NP_001005862.1, and the nucleotide sequence has GeneID2064.

“ErbB3” or “HER3” refers to a receptor tyrosine-protein kinase that isencoded by the human ERBB3 gene and has a role in protein amino acidphosphorylation. The aa sequences for

ErbB3 isoforms are provided at Genbank Accession Nos. NP001973.2 andNP_001005915.1, and the nucleotide sequence has GeneID 2065.

“ErbB4” or “HER4” plays a role in receptor tyrosine kinase signaltransduction that regulates cellular proliferation and differentiation.The aa sequences for ErbB3 isoforms are provided at Genbank AccessionNos. NP001973.2 and NP_001005915.1, and the nucleotide sequence hasGeneID 2066.

The ERBB2, ERBB3, and ERBB4 genes encode heregulin/neuregulin receptors,members of the EGFR-related type I receptor tyrosine kinase subfamily.The encoded proteins form homo- and heterodimers, which complicatesassignment of function: ERBB2 homodimers do not bind heregulin, butERBB2/ERBB3 heterodimers do. Herstatin is a secreted alternative ERBB2product, of the extracellular domain, that binds to p185ERBB2, disruptsERBB2 dimers, reduces p185 phosphorylation, and inhibits growth. HumanERBB2 gene is located at 17p12-21. Overexpression of HER-2 correlateswith poor prognosis in breast carcinoma.

“IGF1R” refers to the insulin-like growth factor 1 receptor. Exemplaryhuman IGF1R nucleic acid and protein sequences are set forth inRefSeqGene Gene ID: 3480 and GenBank Accession Number: NP_000866.1,respectively.

“IGF2R” refers to the insulin-like growth factor 2 receptor. Exemplaryhuman IGF2R nucleic acid and protein sequences are set forth inRefSeqGene Gene ID: 3482 and GenBank Accession Number: NP_000867.2,respectively.

“Insulin receptor” refers to the cellular receptor for insulin.Exemplary human insulin receptor nucleic acid and protein sequences areset forth in RefSeqGene Gene ID: 3643 and GenBank Accession Number:NP_000199.2, respectively.

“RON” refers to the receptor for Macrophage-stimulating proteinreceptor. Exemplary human RON nucleic acid and protein sequences are setforth in RefSeqGene Gene ID: 4486 and GenBank Accession Number:NP_002438.2, respectively.

“c-Kit” refers to v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogenehomolog. Exemplary human c-Kit nucleic acid and protein sequences areset forth in RefSeqGene Gene ID: 3815 and GenBank Accession Number:NP_001087241.1, respectively.

“VEGFR1” refers to vascular endothelial growth factor 1. Exemplary humanVEGFR1 nucleic acid and protein sequences are set forth in RefSeqGeneGene ID: 2321 and GenBank Accession Number: NP_002010.2, respectively.

“VEGFR2” refers to vascular endothelial growth factor 2. Exemplary humanVEGFR2 nucleic acid and protein sequences are set forth in RefSeqGeneGene ID: 3791 and GenBank Accession Number: NP_002244.1, respectively.

“TNFR” refers to tumor necrosis factor receptor. Exemplary human TNFRnucleic acid and protein sequences are set forth in RefSeqGene Gene ID:7132 and GenBank Accession Number: NP_001056.1, respectively.

“FGFR1” refers to fibroblast growth factor receptor 1. Exemplary humanFGFR1 nucleic acid and protein sequences are set forth in RefSeqGeneGene ID: 2260 and GenBank Accession Number: NP_001167537.1,respectively.

“FGFR2” refers to fibroblast growth factor receptor 2. Exemplary humanFGFR2 nucleic acid and protein sequences are set forth in RefSeqGeneGene ID: 2263 and GenBank Accession Number: NP_001138390.1,respectively.

“FGFR3” refers to fibroblast growth factor receptor 3. Exemplary humanFGFR3 nucleic acid and protein sequences are set forth in RefSeqGeneGene ID: 2261 and GenBank Accession Number: NP_000133.1, respectively.

“FGFR4” refers to fibroblast growth factor receptor 4. Exemplary humanFGFR4 nucleic acid and protein sequences are set forth in RefSeqGeneGene ID: 2264 and GenBank Accession Number: NP_075252.2, respectively.

“PDGFR-alpha” refers to platelet-derived growth factor receptor alpha.Exemplary human PDGFR-alpha nucleic acid and protein sequences are setforth in RefSeqGene Gene ID: 5156 and GenBank Accession Number:NP_006197.1, respectively.

“PDGFR-beta” refers to platelet-derived growth factor receptor beta.Exemplary human PDGFR-beta nucleic acid and protein sequences are setforth in RefSeqGene Gene ID: 5159 and GenBank Accession Number:NP_002600.1, respectively.

“EpCAM” refers to epithelial cell adhesion molecule. Exemplary humanEpCAM nucleic acid and protein sequences are set forth in RefSeqGeneGene ID: 4072 and GenBank Accession Number: NP_002345.2, respectively.

“EphA2” refers to EPH receptor A2. Exemplary human EphA2 nucleic acidand protein sequences are set forth in RefSeqGene Gene ID: 1969 andGenBank Accession Number: NP_004422.2, respectively.

“CEA” refers to carcinoembryonic antigen-related cell adhesion molecule5. Exemplary human CEA nucleic acid and protein sequences are set forthin RefSeqGene Gene ID: 1048 and GenBank Accession Number: NP_004354.2,respectively.

“CD44” refers to the cell-surface glycoprotein CD44. Exemplary humanCD44 nucleic acid and protein sequences are set forth in RefSeqGene GeneID: 960 and GenBank Accession Number: NP_001189486.1, respectively.

“ALK” refers to the anaplastic lymphoma receptor tyrosine kinase.Exemplary human

ALK nucleic acid and protein sequences are set forth in RefSeqGene GeneID: 238 and GenBank Accession Number: NP_004295.2, respectively.

“AXL” refers to the AXL receptor tyrosine kinase. Exemplary human AXLnucleic acid and protein sequences are set forth in RefSeqGene Gene ID:558 and GenBank Accession Number: NP_068713.2, respectively.

“EU” indicates that aa positions in a heavy chain constant region,including aa positions in the CH1, hinge, CH2, and CH3 domains, arenumbered herein according to the EU index numbering system (see Kabat etal., in “Sequences of Proteins of Immunological Interest”, U.S. Dept.Health and Human Services, 5^(th) edition, 1991).

“Fab” refers to the antigen binding portion of an antibody, comprisingtwo chains: a first chain that comprises a VH domain and a CH1 domainand a second chain that comprises a VL domain and a CL domain Although aFab is typically described as the N-terminal fragment of an antibodythat was treated with papain and comprises a portion of the hingeregion, it is also used herein as referring to a binding domain whereinthe heavy chain does not comprise a portion of the hinge.

“Fc region” refers to the portion of a single immunoglobulin heavy chainbeginning in the hinge region just upstream of the papain cleavage site(i.e. residue 216 in IgG, taking the first residue of heavy chainconstant region to be 114) and ending at the C-terminus of the antibody.Accordingly, a complete Fc region comprises at least a hinge, a CH2domain, and a CH3 domain. Two Fc regions that are dimerized are referredto as “Fc” or “Fc dimer.” An Fc region may be a naturally occurring Fcregion, or a naturally occurring Fc region in which one or more aas havebeen substituted, added or deleted, provided that the Fc region has thedesired biological properties. A desired biological activity may be anatural biological activity, an enhanced biological activity or areduced biological activity relative to that of the naturally occurringdomain

“Framework region” or “FR” or “FR region” includes the aa residues thatare part of the variable region, but are not part of the CDRs (e.g.,using the Kabat definition of CDRs). Therefore, a variable regionframework is between about 100-120 aas in length but includes only thoseaas outside of the CDRs. For the specific example of a heavy chainvariable region and for the CDRs as defined by Kabat et al., 1991,ibid., framework region 1 corresponds to the domain of the variableregion encompassing aas 1-30; framework region 2 corresponds to thedomain of the variable region encompassing aas 36-49; framework region 3corresponds to the domain of the variable region encompassing aas 66-94,and framework region 4 corresponds to the domain of the variable regionfrom aas 103 to the end of the variable region. The framework regionsfor the light chain are similarly separated by each of the light chainvariable region CDRs. Similarly, using the definition of CDRs by Chothiaet al. or McCallum et al. the framework region boundaries are separatedby the respective CDR termini as described above. In preferredembodiments, the CDRs are as defined by Kabat.

“Full length antibody” or “full length Ab” is an antibody (“Ab”) thatcomprises one or more heavy chains and one or more light chains, whichoptionally may be connected. Each heavy chain is comprised of a heavychain variable region (abbreviated herein as VH) and a heavy chainconstant region. The heavy chain constant region is comprised of threedomains CH1, CH2, and CH3, and optionally a fourth domain, CH4. Eachlight chain is comprised of a light chain variable region (abbreviatedherein as VL) and a light chain constant region. The light chainconstant region is comprised of one domain, CL. The VH and VL regionscan be further subdivided into regions of hypervariability, termedcomplementarity determining regions (CDR), interspersed with regionsthat are more conserved, termed framework regions (FR). Each VH and VLis typically composed of three CDRs and four FRs, arranged fromamino-terminus to carboxyl-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, and FR4 Immunoglobulin proteins can be of any typeor class (e.g., IgG, IgE, IgM, IgD, IgA and IgY) or subclass (e.g.,IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2).

“Gly-Ser linker” or “Gly-Ser peptide” refers to a peptide that consistsof glycine and serine residues. An exemplary Gly-Ser peptide comprisesthe aa sequence (Gly4 Ser)n, wherein n=1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more. In certain embodiments,n is a number between 1 and 5, n is a number between 6 and 10, n is anumber between 11 and 15, n is a number between 16 and 20, n is a numberbetween 21 and 25, or n is a number between 26 and 30.

“Hinge” or “hinge region” or “hinge domain” refers to the flexibleportion of a heavy chain located between the CH1 domain and the CH2domain. It is approximately 25 aas long, and is divided into an “upperhinge,” a “middle hinge” or “core hinge,” and a “lower hinge.” A hingemay be a naturally occurring hinge, or a naturally occurring hinge inwhich one or more aas have been substituted, added or deleted, providedthat the hinge has the desired biological properties. A desiredbiological activity may be a natural biological activity, an enhancedbiological activity or a reduced biological activity relative to thenaturally occurring sequence.

A “hinge subdomain” refers to the upper hinge, middle (or core) hinge orthe lower hinge. The aa sequences of the hinge subdomains of an IgG1,IgG2, IgG3 and IgG4 are set forth in Table 2:

TABLE 2 Listing of IgG hinge subdomains IgG Upper hinge Middle hingeLower hinge Complete hinge IgG1 EPKSCDKTHT CPPCP APELLG SEQ ID NO: 4(SEQ ID NO: 1) (SEQ ID NO: 2) (SEQ ID NO: 3) IgG2 ERKCCVE CPPCP APPVAGPSEQ ID NO: 7 (SEQ ID NO: 5) (SEQ ID NO: 2) (SEQ ID NO: 6) IgG3ELKTPLGDTTHT CPRCP(EPKSCDTPPPCPRCP)₃ APELLG SEQ ID NO: 11 (SEQ ID NO: 8)(SEQ ID NO: 9) (SEQ ID NO: 10) IgG4 ESKYGPP CPSCP APEFLG SEQ ID NO: 15(SEQ ID NO: 12) (SEQ ID NO: 13) (SEQ ID NO: 14)

The complete hinge consists of the upper hinge subdomain, middle hingesubdomain and lower hinge subdomain in amino to carboxy terminal orderand without intervening sequences.

“IC₅₀,” or “IC50” refers to the concentration of a molecule, e.g., aTFcA, that provides a 50% inhibition of a maximal activity (e.g., aresponse to a stimulus or a constitutive activity), i.e., aconcentration that reduces the activity to a level halfway between themaximal activity and the baseline. The IC₅₀ value may be converted to anabsolute inhibition constant (Ki) using, e.g., the Cheng-Prusoffequation. In a system that is inhibited by a binding agent, such as anantibody or a TFcA provided herein, the IC50 may be indistinguishablefrom the EC50.

“Inhibition” of a biological activity by a binding protein refers to anyreproducibly detectable decrease in biological activity mediated by thebinding protein. In some embodiments, inhibition provides astatistically significant decrease in biological activity, e.g., adecrease of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or100% in biological activity relative to the biological activitydetermined in he absence of the binding protein.

“Kabat” in conjunction with designation of immunoglobulin aa sequencepositions indicates that amino acid positions in a light chain constantregion (e.g. CL domain) are numbered according to the Kabat indexnumbering system (see Kabat et al., 1991., op. cit.).

“Linked to” refers to direct or indirect linkage or connection of, incontext, amino acids or nucleotides. An “indirect linkage” refers to alinkage that is mediated through a linker or a domain, comprising, e.g.,one or more aas or nucleotides. A “direct linkage” or “linked directly”when referring to two polypeptide segments refers to the presence ofcovalent bond between the two polypeptide segments, e.g., the twopolypeptide segments are joined contiguously without interveningsequences.

“Linker” refers to one or more aas connecting two domains or regionstogether. A linker may be flexible to allow the domains being connectedby the linker to form a proper three dimensional structure therebyallowing them to have the required biological activity. A linkerconnecting the VH and the VL of an scFv is referred to herein as an“scFv linker” A linker connecting the N-terminus of a VH domain or theC-terminus of the CH3 domain to a second VH or VL domain, e.g., that ofan scFv, is referred to as a “connecting linker”

“Module” refers to a structurally and/or functionally distinct part of aTFcA, such a binding site (e.g., an scFv domain or a Fab domain) and theTFc. Modules provided herein can be rearranged (by recombining sequencesencoding them, either by recombining nucleic acids or by complete orfractional de novo synthesis of new polynucleotides) in numerouscombinations with other modules to produce a wide variety of TFcAs,e.g., as disclosed herein. “Module” is also used to refer to the type ofAEM or DiS modifications. In this context, and as further describedherein, a “module” is one or a combination of two or more aasubstitutions, additions or deletions that are made to enhance or favorthe association or dimerization of the Fc regions comprising thesemodifications.

“% identical” refers to two or more nucleic acid or polypeptidesequences or subsequences that are the same (100% identical) or have aspecified percentage of nucleotide or aa residues that are the same,when the two sequences are aligned for maximum correspondence andcompared. To align for maximum correspondence, gaps may be introducedinto one of the sequences being compared. The aa residues or nucleotidesat corresponding positions are then compared and quantified. When aposition in the first sequence is occupied by the same residue as thecorresponding position in the second sequence, then the sequences areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (e.g., % identity=# of identical positions/total # ofpositions (e.g., overlapping positions)×100). In certain embodiments,the two sequences are the same length. The determination that onesequence is a measured % identical with another sequence can bedetermined using a mathematical algorithm. A non-limiting example of amathematical algorithm utilized for such comparison of two sequences isincorporated in the ALIGN program (version 2.0) which is part of the GCGsequence alignment software package. When utilizing the ALIGN programe.g., for comparing aa sequences, a PAM120 weight residue table, a gaplength penalty of 12, and a gap penalty of 4 may be used. Additionalalgorithms for sequence analysis are well known in the art and many areavailable online

“Portion” or “fragment” (e.g., of a domain) of a reference moiety refersto a discrete part of the whole reference moiety (e.g., domain, e.g., anaturally occurring domain) that is at least, or at most 10% 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% of the size of thereference moiety.

“scFv linker” refers to a peptide or polypeptide domain interposedbetween the VL and VH domains of an scFv. scFv linkers preferably alloworientation of the VL and VH domains in a antigen binding conformation.In one embodiment, an scFv linker comprises or consists of a peptide orpolypeptide linker that only comprises glycines and serines (a “Gly-Serlinker”). In certain embodiments, an scFv linker comprises a disulfidebond.

“Specific binding,” “specifically binds,” “selective binding,” and“selectively binds,” as well as “binds specifically” “bindsselectively,” when referring to the binding of a binding site to itstarget epitope or a combination of binding sites to their targetepitopes, means that the binding site(s) exhibit(s) immunospecificbinding to the target epitope(s). A binding site that binds specificallyto an epitope exhibits appreciable affinity for a target epitope and,generally, does not exhibit cross-reactivity with other epitopes in thatit does not exhibit appreciable affinity to any unrelated epitope andpreferably does not exhibit affinity for any unrelated epitope that isequal to, greater than, or within two orders of magnitude lower than theaffinity for the target epitope. “Appreciable” or preferred bindingincludes binding with a dissociation constant (Kd) of 10⁻⁸, 10⁻⁹ M,10⁻¹⁰, 10⁻¹¹, 10⁻¹² M, 10⁻¹³ M or an even lower Kd value. The Kd valuescan also be indicated as 10e-8, 10e-9 M, etc. Note that lower values forKd (dissociation constant) indicate higher binding affinity, thus a Kdof 10⁻⁷ is a higher Kd value than a Kd of 10⁻⁸, but indicates a lowerbinding affinity than a Kd of 10⁻⁸. Dissociation constants with valuesof about 10⁻⁷ M, and even as low as about 10⁻⁸M, are at the high end ofdissociation constants suitable for therapeutic antibodies. Bindingaffinities may be indicated by a range of dissociation constants, forexample, 10⁻⁶ to 10⁻¹² M, 10⁻⁷ to 10⁻¹² M, 10⁻⁸ to 10⁻¹² M or better(i.e., or lower value dissociation constant). Dissociation constants inthe nanomolar (10⁻⁹ M) to picomolar (10⁻¹² M) range or lower aretypically most useful for therapeutic antibodies. Suitable dissociationconstants are Kds of 50 nM or less (i.e., a binding affinity of 50 nM orhigher—e.g., a Kd of 45 nM) or Kds of 40 nM, 30 nM, 20 nM, 10 nM, 1 nM,100 pM, 10 pM or 1 pM or less. Specific or selective binding can bedetermined according to any art-recognized means for determining suchbinding, including, for example, according to Scatchard analysis and/orcompetitive binding assays.

A “TFc” or “tandem Fc” refers to an entity comprising in an amino tocarboxyl terminal order: a first Fc region, which is linked at itsC-terminus to the N-terminus of a TFc linker, which is linked at itsC-terminus to the N-terminus of a second Fc region, wherein the firstand the second Fc regions associate to form an Fc.

“TFcA” refers to a tandem Fc antibody. A TFcA may be a monovalent ormonospecific TFcA, e.g., comprising a single binding site. A TFcA mayalso be a bispecific TFcA, which is referred to herein as a TFcBA. ATFcA may be monoclonal.

“TFcBA” refers to a tandem Fc bispecific antibody, an artificial hybridprotein comprising at least two different binding moieties or domainsand thus at least two different binding sites (e.g., two differentantibody binding sites), wherein one or more of the pluralities of thebinding sites are covalently linked, e.g., via peptide bonds, to eachother. An exemplary TFcBA described herein is an anti-c-Met+anti-EGFRTFcBA, which is a polyvalent bispecific antibody that comprises a firstbinding site binding specifically to a c-Met protein, e.g., a humanc-Met protein, and one or more second binding sites binding specificallyto an EGFR protein, e.g., a human EGFR protein. When a TFcBA namecomprises two antigens separated by a plus sign (+) this indicates thatthe binding sites for the two antigens may be in either relative aminoto carboxy orientation in the molecule, whereas when the TFcBA namecomprises two antigen binding site names separated by a slash (/) theantigen binding site to the left of the slash is amino terminal to theantigen binding site to the right of the slash. A TFcBA may be abivalent binding protein, a trivalent binding protein, a tetravalentbinding protein or a binding protein with more than 4 binding sites. Anexemplary TFcBA is a bivalent bispecific antibody, i.e., an antibodythat has 2 binding sites, each binding to a different antigen orepitope. In certain embodiments, the N-terminal binding site of a TFcBAis a Fab and the C-terminal binding site is an scFv.

Tandem Fc Abs

Provided herein are Tandem Fc Antibodies (“TFcAs”), which may bemonovalent or polyvalent, e.g., bivalent, trivalent, or tetravalent.TFcAs which are polyvalent may be monospecific, bispecific (“Tandem FcBispecific Abs” or “TFcBAs”) trispecific or tetraspecific TFcBAs. When aTFcBA is multispecific, it may be monovalent for one or morespecificities.

In certain embodiments, a TFcA is a TFcBA. Exemplary TFcBAs inhibitligand-induced signal transduction through one or both of the receptorstargeted by the TFcBA and may thereby inhibit tumor cell proliferationor tumor growth. TFcBAs may also induce receptor downregulation or blockreceptor dimerization. Exemplary anti c-Met/anti-EGFR TFcBAs comprise asingle anti-c-Met binding site (monovalent for anti-c-Met) and one ormore anti-EGFR binding sites (monovalent or polyvalent for anti-EGFR). ATFc typically comprises a first Fc region linked to a second Fc regionthrough a TFc linker, wherein the first and the second Fc regionsdimerize to form an Fc.

FIG. 1 shows a diagram of an exemplary TFcBA showing the variouselements of the molecule. As shown in the Figure, a TFcBA comprises afirst binding site (e.g., an anti-c-Met Fab), a second binding site(e.g., an anti-EGFR scFv), and a tandem Fc (“TFc”) that links the firstand the second binding sites together. A TFcBA may be described ascontaining three modules, wherein the first module comprises the firstbinding site, the second module comprises the TFc and the third modulecomprises the second binding site. A TFc generally comprises in acontiguous aa sequence a first Fc region, a TFc linker, and a second Fcregion, wherein the TFc linker links the first Fc region to the secondFc region and allows the association of the two Fc regions. Asillustrated in the exemplary TFcBA in FIG. 1, each of the two Fc regionsof a TFc may comprise a hinge, a CH2 domain and a CH3 domain Each ofthese regions may be from the same immunoglobulin isotype, or fromdifferent isotypes. For example, the hinge, CH2 and CH3 domains may allbe from IgG1, IgG2, IgG3 or IgG4, or certain domains or portions thereofmay be from one immunoglobulin isotype and another domain or portion maybe from another immunoglobulin isotype. For example, the TFcBA that ispictured in FIG. 1 comprises all domains from IgG1, or alternatively, itmay comprise an IgG1/IgG4 hybrid hinge, an IgG4 CH2 domain and an IgG1CH3 domain. An Fc region preferably comprises human Fc domains, however,sequences from other mammals or animals may also be used, provided thatthe TFcBA retains its biological activity and is preferably notsignificantly immunogenic in a human subject.

In preferred embodiments, the first and/or the second Fc region compriseone or more modifications to enhance their association and/or tostabilize such association. In certain embodiments, the first and/or thesecond CH3 domains of a TFcA comprises one or more modification toenhance the association of the CH3 domains or Fcs comprising such. Suchmodifications are referred to herein as Association EnhancingModifications or “AEMs.” Exemplary modifications include aasubstitutions in both CH3 domains to enhance their interaction, e.g.,knob/hole mutations.

In certain embodiments, the first and/or the second Fc region comprisesan aa modification that results in the addition of one or more cysteinesto the Fc region, to thereby form a disulfide bond with the other Fcregion of the TFc. Such modifications are referred to herein asdisulfide forming modifications or “DiS” modifications. DiSmodifications may be present in the hinge, CH2 and/or CH3 domains.

A TFc may comprise one or more AEM and/or one or more DiS modifications.FIG. 1B shows exemplary modifications that can be made to either the CH3region or the hinge. Fc regions may also comprise additionalmodifications, e.g., modifications that modulate a biological activitythat is mediated through the Fc region, such as ADCC.

Although generally, the first and the second Fc regions comprise ahinge, a CH2 domain and a CH3 domain, in certain embodiments, an Fcregion may comprise a CH3 domain and a CH2 domain, but no hinge. Inother embodiments, an Fc region comprises a CH3 domain and a hinge, butdoes not comprise a CH2 domain. In other embodiments, an Fc region maycomprise a CH3 domain and a CH4 domain, but does not comprise a CH2domain nor a hinge. In other embodiments, an Fc region may comprise aCH3 domain, a CH4 domain, a CH2 domain, but does not comprise a hinge.In other embodiments, an Fc region may comprise a CH3 domain, a CH4domain, a hinge, but does not comprise a CH2 domain. In certainembodiments, a portion of one or more domains is absent.

In certain embodiments, the first Fc region comprises an aa sequencethat differs from that of the second Fc region in one or more aaaddition, deletion or substitution (a “heterodimeric Fc”). This is oftenthe case as AEM and DiS modifications, which typically introducedifferent modifications to the first and the second Fc region. In otherembodiments, the first Fc region comprises the same aa sequence as thesecond Fc region (a “homodimeric Fc”).

In certain embodiments, an Fc domain (hinge, CH2 or CH3 domain) isdirectly linked to another Fc domain. For example, a hinge may bedirectly linked to a CH2 domain and/or a CH2 domain may be directlylinked to a CH3 domain. In other embodiments, an Fc domain is linked toanother Fc domain through a linker, which may be one or more aas long,provided that the TFcA comprising these domains has the desiredbiological activity and stability and any other desired characteristics.

In certain embodiments, a binding site is an antigen binding site, whichcomprises, e.g., a heavy chain variable (VH) domain and a light chainvariable (VL) domain. The VH and VL domains generally contain 3Complementarity Determining Regions (CDRs) each, although in certainembodiments, fewer than 6 CDRs may be sufficient for providing specificbinding to an antigen. In certain embodiments, the VH domain is part ofa Fab, in which case, the VH domain is linked to a CH1 domain, generallyin the natural order, i.e., the VH domain is linked to the N-terminus ofthe CH1. When the antigen binding site is part of a Fab, the VL domainmay be linked to a light chain constant (CL) domain, generally in thenatural order, i.e., the VL domain is linked to the N-terminus of the CLdomain.

The variable domains (VH and VL) may be linked directly or indirectly tothe constant domains (CH1 and CL), e.g., through a linker, which may beone or more aas long, provided that the TFcA comprising these domainshas the desired biological activity and stability and any other desiredcharacteristics.

In certain embodiments, the VH domain is part of an scFv, in which case,the VH domain is linked to the VL domain through an scFv linker, and thescFv is linked to the N- and/or C-terminus of a TFc. When a binding siteis an scFv, the variable regions are generally not linked to a CH1 or CLdomain

In certain embodiments, a TFcA is monovalent and monospecific. Amonovalent TFcA may comprise a binding site at the amino terminus or atthe C-terminus of the TFc. The binding site of a monovalent TFcA may bea Fab or an scFv. Exemplary heavy chains of monovalent TFcAs comprise inamino to carboxyl terminal order:

-   -   i) a VH domain and a TFc;    -   ii) a VH domain, a CH1 domain, and a TFc;    -   iii) a VH domain, an scFv linker, a VL domain, and a TFc;    -   iv) a TFc, a connecting linker and a VH domain;    -   v) a TFc, a connecting linker, a VH domain and a CH1 domain; and    -   vi) a TFc, a connecting linker, a VH domain, an scFv linker and        a VL domain.

When a TFcA comprises a Fab, the TFcA also comprises a light chaincomprising the VL domain of the Fab and optionally a CL domain

In certain embodiments, a TFcA is a TFcBA. TFcBAs may comprise one Fabbinding specifically to a first antigen and a second Fab bindingspecifically to a second antigen. TFcBAs may also comprise a first scFvbinding specifically to a first antigen and a second scFv bindingspecifically to a second antigen. TFcBAs may also comprise a Fab bindingspecifically to a first antigen and an scFv binding specifically to asecond antigen. In certain embodiments, the amino terminus of a TFc isconnected to a Fab and the carboxyl terminus of the TFc is connected toan scFv. Alternatively, the amino terminus of a TFc is connected to anscFv and the carboxyl terminus of the TFc is connected to a Fab.Exemplary molecules have the following format: Fab-TFc-scFv;Fab-TFc-Fab; scFv-TFc-scFv; and scFv-TFc-Fab.

In one embodiment, a TFcBA comprises a heavy chain, which comprises inamino to carboxyl-terminal order:

-   -   (i) a first VH domain, a TFc, a connecting linker, and a second        VH domain;    -   (ii) a first VH domain, a CH1 domain, a TFc, a connecting        linker, and a second VH domain;    -   (iii) a first VH domain, a CH1 domain, a TFc, a connecting        linker, a second VH domain, an scFv linker and a second VL        domain, wherein the second VH and VL domains associate to form a        second binding site;    -   (iv) a first VH domain, a TFc, a connecting linker, a second VH        domain, and a CH1 domain;    -   (v) a first VH domain, a first CH1 domain, a TFc, a connecting        linker, a second VH domain and a second CH1 domain;    -   (vi) a first VH domain, a first scFv linker, a first VL domain,        a TFc, a connecting linker, and a second VH domain, wherein the        first VL and VH domains associate to form a first binding site;    -   (vii) a first VH domain, a first scFv linker, a first VL domain,        a TFc, a connecting linker, a second VH domain, and a CH1        domain, wherein the first VL and VH domains associate to form a        first binding site; and    -   (viii) a first VH domain, a first scFv linker, a first VL        domain, a TFc, a connecting linker, a second VH domain, a second        scFv linker, and a second VL domain, wherein the first VH and VL        domains form a first binding site and the second VH and VL        domains form a second binding site.

A TFcBA of (i-)(v) may further comprise a light chain comprising a firstVL domain and optionally a CL domain located at the C-terminus of the VLdomain, wherein the first VH and VL domains associate to form a firstbinding site. A TFcBA of (i), (ii), (iv)-(vii) may comprise a lightchain comprising a second VL domain and optionally a CL domain locatedat the C-terminus of the VL domain, wherein the first VH and VL domainsassociate to form a second binding site.

In certain embodiments, a heavy chain comprises a first VH domain, whichis linked at its C-terminus to the N-terminus of a TFc, which is linkedat its C-terminus to the N-terminus of a connecting linker, which islinked at its C-terminus to the N-terminus of a second VH domain. Incertain embodiments, a heavy chain comprises a first VH domain, which islinked at its C-terminus to the N-terminus of a CH1 domain, which islinked at its C-terminus to the N-terminus of a TFc, which is linked atits C-terminus to the N-terminus of a connecting linker, which is linkedat its C-terminus to the N-terminus of a second VH domain. In certainembodiments, a heavy chain comprises a first VH domain, which is linkedat its C-terminus to the N-terminus of a CH1 domain, which is linked atits C-terminus to the N-terminus of a TFc, which is linked at itsC-terminus to the N-terminus of a connecting linker, which is linked atits C-terminus to the N-terminus of a second VH domain, which is linkedat its C-terminus to the N-terminus of an scFv linker, which is linkedat its C-terminus to the N-terminus of a second VL domain, wherein thesecond VH and VL domains associate to form a second binding site. Incertain embodiments, a heavy chain comprises a first VH domain, which islinked at its C-terminus to the N-terminus of a TFc, which is linked atits C-terminus to the N-terminus of a connecting linker, which is linkedat its C-terminus to the N-terminus of a second VH domain, which islinked at its C-terminus to the N-terminus of a CH1 domain. In certainembodiments, a heavy chain comprises a first VH domain, which is linkedat its C-terminus to the N-terminus of a first CH1 domain, which islinked at its C-terminus to the N-terminus of a TFc, which is linked atits C-terminus to the N-terminus of a connecting linker, which is linkedat its C-terminus to the N-terminus of a second VH domain, which islinked at its C-terminus to the N-terminus of a second CH1 domain. Incertain embodiments, a heavy chain comprises a first VH domain, which islinked at its C-terminus to the N-terminus of a first scFv linker, whichis linked at its C-terminus to the N-terminus of a first VL domain,which is linked at its C-terminus to the N-terminus of a TFc, which islinked at its C-terminus to the N-terminus of a connecting linker, whichis linked at its C-terminus to the N-terminus of a second VH domain,wherein the first VH and VL domains associate to form a first bindingsite. In certain embodiments, a heavy chain comprises a first VH domain,which is linked at its C-terminus to the N-terminus of a first scFvlinker, which is linked at its C-terminus to the N-terminus of a firstVL domain, which is linked at its C-terminus to the N-terminus of a TFc,which is linked at its C-terminus to the N-terminus of a connectinglinker, which is linked at its C-terminus to the N-terminus of a secondVH domain, which is linked at its C-terminus to the N-terminus of a CH1domain, wherein the first VH and VL domains associate to form a firstbinding site. In certain embodiments, a heavy chain comprises a first VHdomain, which is linked at its C-terminus to the N-terminus of a firstscFv linker, which is linked at its C-terminus to the N-terminus of afirst VL domain, which is linked at its C-terminus to the N-terminus ofa TFc, which is linked at its C-terminus to the N-terminus of aconnecting linker, which is linked at its C-terminus to the N-terminusof a second VH domain, which is linked at its C-terminus to theN-terminus of a second scFv linker, which is linked at its C-terminus tothe N-terminus of a second VL domain, wherein the first VH and VLdomains form a first binding site and the second VH and VL domains forma second binding site.

In certain embodiments, a VL domain is substituted for the VH domain andthe VH domain is substituted for the VL domain in the constructs above.

When the heavy chain does not comprise either a first or a second VLdomain, a VL domain may be provided by a light chain. A light chain maycomprise a first or a second VL domain and optionally a CL domain. Forexample, an scFv may comprise in amino to carboxy terminal order a VLdomain, an scFv linker and a VH domain.

In certain embodiments, a TFcBA comprises a first antigen binding sitethat binds specifically to a first receptor and a second antigen bindingsite that binds specifically to a second receptor. In certainembodiments, the first antigen binding site that binds specifically tothe first receptor is a Fab and the second antigen binding site thatbinds specifically to the second receptor is an scFv. Exemplarycombinations of binding sites are set forth in Table 3, wherein a “yes”indicates a possible combination and anti-c-Met+anti-EGFR TFcBAs areused to illustrate the possible combinations:

TABLE 3 Exemplary combinations of binding sites of anti-c-Met +anti-EGFR TFcBAs Binding site linked to the N-terminus of the TFcAnti-c-Met Anti-c-Met Anti-EGFR Anti-EGFR scFv Fab scFv Fab Binding sitelinked Anti-c-Met scFv yes yes yes yes to the C-terminus Anti-c-Met Fabyes yes yes yes of the TFc Anti-EGFR scFv yes yes yes yes Anti-EGFR Fabyes yes yes yes

In certain embodiments, a TFcBA comprises more than 2 binding sites. ATFcBA may comprise 3, 4, 5, 6 or more binding sites. Additional bindingsites may be linked, e.g., to the N- and/or C-terminus of a TFcA orTFcBA. For example, a heavy chain may comprise one or more Fabs and/orscFvs linked to the amino- or carboxyl-terminus of the TFc.

Exemplary domains of TFcBAs are further described below.

Exemplary Hinges

In one embodiment, the first and/or the second Fc region of a TFcA,e.g., a TFcBA, comprises an IgG upper hinge, an IgG middle hinge and/oran IgG lower hinge. For example, an Fc region may comprise one or moreIgG1 upper, middle and lower hinge, e.g., set forth in SEQ ID NOs:1, 2and 3, respectively (see Table 2). Fc regions may also comprise one ormore of an IgG2 upper, middle and lower hinge, e.g., set forth in SEQ IDNOs:5, 2 and 6, respectively (the middle hinge of IgG1 and IgG2 have thesame aa sequence/see Table 2). Fc regions may also comprise one or moreof an IgG3 upper, middle and lower hinge, e.g., set forth in SEQ IDNOs:8, 9 and 10, respectively (Table 2). Fc regions may also compriseone or more of an IgG4 upper, middle and lower hinge, e.g., set forth inSEQ ID NOs:12, 13 and 14, respectively (Table 2). Fc regions may alsocomprise one or more mouse Ig sequences or IgA1 or IgA2 sequences.

A first and/or a second Fc region of a TFcA may also comprise an aasequence of an upper, middle, or lower hinge having an aa sequence thatdiffers from a naturally occurring sequence, such as a sequence setforth herein (e.g., SEQ ID NOs:1-14) comprising up to 1, 2, 3, 4, or 5aa modifications, e.g., aa substitutions, deletions or additions. Forexample, the following IgG1 upper hinges may be used:

EPKSCDKTCC (SEQ ID NO:16; corresponds to SEQ ID NO:1 with the aasubstitutions H224C and T225C (underlined)) and

EPKSCDKCHT (SEQ ID NO:17; corresponds to SEQ ID NO:1 with the aasubstitution T223C (underlined)).

The amino acid numbering of the hinge residues referred to herein isaccording to their numbering in a full length antibody (EU numbering;see FIG. 2).

In one embodiment, the first and/or the second hinge of a TFcA is a fulllength wild type IgG1 hinge comprising the following aa sequence:

(SEQ ID NO: 4) EPKSCDKTHTCPPCPAPELLG.

The first and/or the second hinge of a TFcBA may also consist of an IgG1hinge comprising up to 1, 2, 3, 4, or 5 aa modifications, e.g., aasubstitutions, deletions or additions, relative to SEQ ID NO:4. Forexample, the following IgG1 hinges may be used:

EPKSCDKTCCCPPCPAPELLG (SEQ ID NO:18; corresponds to SEQ ID NO:4 with theaa substitutions H224C and T225C); and

EPKSCDKCHTCPPCPAPELLG (SEQ ID NO:19; corresponds to SEQ ID NO: 4 withthe aa substitution T223C).

In one embodiment, the first and/or the second hinge of a TFcA is ahybrid hinge, i.e., a hinge that comprises portions from different IgGsubclasses. In one embodiment, a hinge comprises an upper hinge fromIgG1 and a middle and lower hinge from IgG4, and may, e.g., consist ofthe following aa sequence:

EPKSCDKTHTcpscpapeflg (SEQ ID NO:20; upper case residues represents IgG1sequences and lower case residues represent IgG4 sequences).

The first and/or the second hinge of a TFcBA may also be a hybrid hingecomprising the aa sequence set forth in SEQ ID NO:20, comprising up to1, 2, 3, 4, or 5 aa modifications, e.g., aa substitutions, deletions oradditions. For example, the following IgG1/IgG4 hybrid hinges may beused:

EPKSCDKTCCcpscpapeflg (SEQ ID NO:21; corresponds to SEQ ID NO:20 withthe aa substitutions H224C and T225C; upper case residues represent IgG1sequences and lower case residues represent IgG4 sequences); and

EPKSCDKCHTcpscpapeflg (SEQ ID NO:22; corresponds to SEQ ID NO:20 withthe aa substitution T223C).

In certain embodiments, the first and/or the second Fc region comprisesa portion of a hinge instead of a full length hinge. For example, afirst and/or a second Fc region of a TFcBA may comprise a hinge lackingthe upper, middle and/or lower hinge. In certain embodiments, an Fcregion comprises a middle and lower hinge, but does not comprise anupper hinge. An exemplary aa sequence of an IgG1 middle and lower hingeis the following:

(SEQ ID NO: 23) CPPCPAPELLG.

An exemplary aa sequence of an IgG4 middle and lower hinge is thefollowing:

(SEQ ID NO: 24) CPSCPAPEFLG.

A summary of the aa numbers of the hinges and portions thereof providedabove is set forth in Table 4. Alignments of the IgG1 and IgG1/IgG4hybrid hinges are set forth in FIG. 2.

TABLE 4 SEQ ID NOs of exemplary hinges and subdomains thereof IgG1/IG4IgG1 IgG4 hybrid hinge Upper hinge SEQ ID NO: 1 SEQ ID NO: 12 SEQ ID NO:1 Upper hinge with SEQ ID NO: 16 — SEQ ID NO: 16 H224C/T225C Upper hingewith SEQ ID NO: 17 — SEQ ID NO: 17 T223C Middle hinge SEQ ID NO: 2 SEQID NO: 13 SEQ ID NO: 13 Lower hinge SEQ ID NO: 3 SEQ ID NO: 14 SEQ IDNO: 14 Middle and lower SEQ ID NO: 23 SEQ ID NO: 24 SEQ ID NO: 24 hingeComplete hinge SEQ ID NO: 4 SEQ ID NO: 15 SEQ ID NO: 20 Complete hingeSEQ ID NO: 18 — SEQ ID NO: 21 with H224C/T225C Complete hinge SEQ ID NO:19 — SEQ ID NO: 22 with T223C

Cysteines may also be introduced at positions other than T223, H224 andT225 in a hinge, e.g., by the substitution K222C, described inWO2010/064090.

Additional hinges that may be used in TFcAs include hIgG1 hinge variantscomprising one of the following aa sequences (FIG. 2):

(SEQ ID NO: 263; hIgG1 Extra Prolines v1) PPPPCDKTHTCPPCP(SEQ ID NO: 264; hIgG1 Extra Prolines v2) EPKSCPPPCPPCP(SEQ ID NO: 265; hIgGl-like double core) EPKSCPPCPCPPCP

Hinges that may be used in TFcAs may also include mouse hinge sequences,e.g., mIgG1 and mIgG2 sequences, and hybrids thereof. An exemplarymIgG1/mIgG2A hinge comprises the aa sequence VPRDCTIKPCPPCP (SEQ IDNO:267).

Other hinges that may be used in TFcAs comprise an IgG2 hinge or variantthereof, such as a variant comprising one of the following amino acidsequences (FIG. 2):

(SEQ ID NO: 268; hIgG2 C232P) ERKPCVECPPCP (SEQ ID NO: 269; hIgG2 C233P)ERKCPVECPPCP

In certain embodiments, a TFcA comprises an IgA, e.g., IgA2, hinge orvariant thereof. Exemplary IgA2 hinge variants include those comprisingone of the following aa sequences (FIG. 2):

(SEQ ID NO: 271; hIgA2 Modified v1) EPKSCPCPPPPPCCP(SEQ ID NO: 272; hIgA2 modified v2) EPKSCPCPPPPCCP(SEQ ID NO: 273; hIgA2 Modified v3) EPKSCPVPPPPPCCP

Other variations, e.g., aa modifications, may also be introduced into ahinge. For example, the substitution S228P may be made in the middlehinge of IgG4 to stabilize the interaction between two Fc regionscomprising IgG4 middle hinges.

A TFcA comprising IgG2 sequences in its Fab domain may comprise themutation C129S in the heavy chain portion of the Fab domain, which is amutation of the cysteine that normally links the heavy chain to thelight chain. Such a mutation will encourage the formation of a disulfidebridge between the light chain cysteine and C232 in the heavy chain, andC233 will pair with C233 of the neighboring hinge (in addition to thetwo disulfides in the CPPCP motif).

In certain embodiments, the following variant hinges are used:PRDCGCKPCICT (SEQ ID NO:248), PKSCGCKPCICT (SEQ ID NO:249), PKSCGCKPCICP(SEQ ID NO:250), PRDCGCKPCPPCP (SEQ ID NO:251), PRDCGCHTCPPCP (SEQ IDNO:252), PKSCDCHCPPCP (SEQ ID NO:253), and RKCCVECPPCP (SEQ ID NO:254).

In certain embodiments, a TFcBA does not comprise a first or a secondhinge. For example, instead of a first hinge, a TFcBA may comprise aconnecting linker that links the first binding site to the first CH2domain. Such a linker may be a Gly-Ser linker as further describedherein in the context of TFc linkers. In certain embodiments, aconnecting linker comprises a (G₄S)₂ or (G₄S)₃ or (G₄S)₄ sequence. Otherpeptide sequences may also be used as a connecting linker provided thatthey provide the required flexibility and rigidity of certain parts ofthe linker. In certain embodiments, a TFcBA does not comprise a secondhinge, but comprises a connecting linker instead, which may be a Gly-Serlinker similar to that of the TFc linker.

Exemplary CH2 Domains

In certain embodiments, an Fc region comprises a CH2 domain. A CH2domain may be from a human IgG1, IgG2, IgG3 or IgG4 or from acombination thereof (a “hybrid” CH2 domain). An exemplary full lengthwild type IgG1 CH2 domain consists of the following aa sequence:

(SEQ ID NO: 261) GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAK.

An exemplary full length IgG1 CH2 domain with an N297Q substitution toreduce the glycosylation at that residue, such that the variant issubstantially aglycosylated when expressed in a mammalian cell, consistsof the following amino acid sequence.

(SEQ ID NO: 25) GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAK.

An exemplary full length wild type IgG4 CH2 domain comprises thefollowing aa sequence:

(SEQ ID NO: 262) GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AK.

An exemplary full length IgG4 CH2 domain with a T299K substitution toreduce the glycosylation at residue 297, such that the variant issubstantially aglycosylated when expressed in a mammalian cell, consistsof the following amino acid sequence:

(SEQ ID NO: 26) GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSKYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAK.

A CH2 domain may also comprise an aa sequence that differs from that ofIgG1, IgG2, IgG3 or IgG4 in one or more aa modifications, e.g., aadeletions, additions or substitutions. In certain embodiments, a CH2domain comprises an aa sequence that differs from that of a naturallyoccurring (or wild type) CH2 domain (e.g., SEQ ID NO:261 and 262) orfrom SEQ ID NOs:25 or 26 in at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25 or 30 aas. In certain embodiments, a CH2 domain comprises an aasequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99%identical or similar to that of a naturally occurring CH2 domain (e.g.,SEQ ID NO:261 and 262) or SEQ ID NOs:25 or 26. Exemplary modificationsinclude other modifications to reduce or remove glycosylation at aa 297.A modification may generally comprise an amino acid substitution in anyof EU positions 297-299 (aa motif NXT) such that the variant issubstantially aglycosylated when expressed in a mammalian cell. Inaddition to T299K, other substitutions that may be made at aa 299 toreduce glycosylation at aa 297 include T299S, T299A, T299N, T299G,T299Y, T299C, T299H, T299E, T299D, T299R, T299G, T299I, T299L, T299M,T299F, T299P, T299W, and T299V, as described, e.g., in WO/2005/018572.

Other aa changes may affect antibody effector functions, e.g., ADCC andCDC, or stability or other desired antibody characteristic. For example,FcγRI binding to an IgG1 Fc region may be modulated by modifying Leu235and/or Gly237. Binding to C1q for CDC may be modulated by substitutionof Ala330 and/or Pro331. Other modifications that may be made to CH2domains to modulate the effector functions include substitutions at oneor more aas at positions 234 to 238, 253, 279, 310, 318, 320, and 322.

Exemplary CH3 Domains

In certain embodiments, the first and/or the second Fc region of a TFcA,e.g., a TFcBA, comprises a CH3 domain. A CH3 domain may be from a humanimmunoglobulin, e.g., an IgG1, IgG2, IgG3 or IgG4, or from a combinationthereof (a “hybrid” CH3 domain). An exemplary full length wild type IgG1CH3 domain comprises the following aa sequence:

(SEQ ID NO: 27) GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK.

In certain embodiments, variations of SEQ ID NO:27 may be used. Forexample, the C-terminal lysine of the CH3 domain may be deleted (see SEQID NO:28 in FIG. 3). In other embodiments, a CH3 domain comprises the aasubstitutions D356E and L358M and the C-terminal lysine may be presentor absent (SEQ ID Nos:29 and 30 respectively, and shown in FIG. 3).

In certain embodiments, the first and/or the second CH3 domains of aTFcA are modified to enhance the association of the first and the secondFc comprising the first and the second CH3 domains, respectively. SuchCH3 modifications are referred to herein as Association EnhancingModifications (“AEMs”). As further described in the Examples, it hasbeen unexpectedly found that the addition of a TFc linker joining thetwo Fc regions of an Ab further enhances the ability of Abs having AEMsto properly assemble and increases their stability.

Exemplary AEM modifications that can be used are modifications thatcreate “knobs-into-holes” and which are described, e.g., in U.S. Pat.No. 7,183,076. In this strategy, the CH3 domains are engineered to giveone a protruding “knob” or “bump” and the other a complementary “hole,”thereby favoring the association of the CH3 domains. An exemplary aamodification to a CH3 domain that creates a “hole” is the combination ofaa substitutions T366S, L368A and Y407V (e.g., in SEQ ID NOs:31-34; FIG.3). Such a CH3 domain with a “hole” will dimerize favorably with a CH3domain having a “knob” or “bump,” e.g., a CH3 domain comprising theamino acid substitution T366W (e.g., in SEQ ID NOs:35-38; FIG. 3). Thispair of knob/hole mutations is referred to herein as “AEM module 1” or“AEM 1,” of which the first and the second CH3 domains are referred toas “AEM 1.1” and “AEM 1.2,” respectively.

In another embodiment, one of the two CH3 domains of a TFcA comprises ahole created by the substitution Y407T (e.g., in SEQ ID NOs:39-42; FIG.3) and the other CH3 domain comprises a knob created by the substitutionT366Y (e.g., in SEQ ID NOs:43-46; FIG. 3). This second pair of knob/holemutations is referred to as “AEM module 2” or “AEM 2,” of which thefirst and the second CH3 domains are referred to as “AEM 2.1” and “AEM2.2,” respectively.

The association between two CH3 domains may also be enhanced bymechanisms other than those creating typical knob/holes, e.g., byelectrostatic modifications. In one embodiment, one of the two CH3domains of a TFcA comprises the combination of substitutions S364H andF405A (e.g., in SEQ ID NOs:47-50; FIG. 3) and the other CH3 domaincomprises the combination of substitutions Y349T and T394F (e.g., in SEQID NOs:51-54; FIG. 3). This third pair of modifications is referred toherein as “AEM module 3” or “AEM 3,” of which the first and the secondCH3 domains are referred to as “AEM 3.1” and “AEM 3.2,” respectively.

In one embodiment, one of the two CH3 domains of a TFcA comprises thecombination of substitutions K370D, K392D and K409D (e.g., in SEQ IDNOs:55-58; FIG. 3) and the other CH3 domain comprises the combination ofsubstitutions E(or D)356K, E357K and D399K (e.g., in SEQ ID NOs:59-62;FIG. 3). This fourth pair of modifications is referred to herein as “AEMmodule 4” or “AEM 4,” of which the first and the second CH3 domains arereferred to as “AEM 4.1” and “AEM 4.2,” respectively. The aa at position356 may be either an E or a D, depending on the sequence that is used,and therefore the substitution at that position is referred to as “E (orD)356.”

In certain embodiments, the first and/or the second CH3 domains of aTFcA comprise one or more aa modifications resulting in the addition ofone or more Cysteines that allow the formation of one or more disulfidebonds between the two CH3 or Fc domains. In one embodiment, one of thetwo CH3 domains of a TFcA comprises the substitution Y349C (e.g., in SEQID NOs:63-66; FIG. 3) and the other CH3 domain comprises thesubstitution S354C (e.g., in SEQ ID NOs:67-70; FIG. 3). This pair ofdisulfide forming modifications is referred to herein as “DiS module 1”or “DiS 1,” of which the first and the second CH3 domains are referredto as “DiS 1.1” and “DiS 1.2,” respectively.

In other embodiments, a cysteine is added to the C-terminus of each ofthe two CH3 domains of a TFcA, to thereby form a disulfide bond betweenthe two CH3 domains. For example, one of the two CH3 domains maycomprise the substitution of the carboxyl terminal aas “PGK” with“KSCDKT” (e.g., in SEQ ID NOs:71-72; FIG. 3) and the other CH3 domainmay comprise the substitution of the carboxyl-terminal aas “PGK” with“GEC” (e.g., in SEQ ID NOs:73-74; FIG. 3).

In certain embodiments, a CH3 domain comprises a combination of two ormore aa change(s). For example, one or more AEMs may be combined withone or more DiS modifications. In an exemplary embodiment, a CH3 domaincomprises the hole mutations T366S, L368A, Y407V and the disulfide bondgenerating mutation Y349C (AEM 1.1+DiS 1.1). Such a CH3 domain may becombined in a TFc with a CH3 domain comprising the knob mutation T366Wand the disulfide bond generating mutation S354C (AEM 1.2+DiS 1.2).Exemplary aa sequences comprising this combination of substitutionsinclude SEQ ID NOs:75-82 (FIG. 3).

Exemplary combinations of AEMs and DiSs that are made in CH3 domains tofavor the association of CH3 domains or Fc regions comprising these areset forth in Table 5, wherein a “yes” indicates a combination that maybe used.

TABLE 5 Exemplary combinations of AEM and DiS modifications AEM module 1AEM module 4 1.1 AEM module 3 4.1 4.2 T366S/ AEM module 2 3.1 3.2 K370D/D356K*/ L368A/ 1.2 2.1 2.2 S364H/ Y349T/ K392D/ E357K/ Y470V T366W Y407TT366Y F405A T394F K409D D399K DiS 1 1.1 Y349C yes yes yes yes yes yesyes yes 1.2 S354C yes yes yes yes yes yes yes yes DiS 2 2.1 C. term. yesyes yes yes yes yes yes yes KSCDKT 2.2 C term. yes yes yes yes yes yesyes yes GEC *With respect to the sequences that have an E at position356 (e.g., SEQ ID NOs: 29 and 30), this mutation is E356K.

Aa sequences of exemplary IgG1 CH3 domains with an AEM and/or DiScomprise SEQ ID NOs:31-98. An alignment of these aa sequences isprovided in FIG. 3, and a description of these sequences is provided inTable 6. The CH3 domains in Table 6 and FIG. 3 are organized accordingto their AEM module (number 1, 2, 3 or 4) and their DiS module (number 1or 2). Compatible CH3 domains are listed as “1” and “2” preceded by themodule number.

TABLE 6 SEQ ID NOs for IgG1 CH3 domains with an AEM and/or a DiS Wildtype with D356E Wild type Wild type and L358M; without with D356Ewithout a Wild type terminal lysine and L358M terminal lysine — SEQ IDSEQ ID NO: 28 SEQ ID NO: 29 SEQ ID NO: 30 NO: 27 AEM AEM 1.1T366S/L368A/ SEQ ID SEQ ID NO: 32 SEQ ID NO: 33 SEQ ID NO: 34 1 Y407VNO: 31 AEM 1.2 SEQ ID SEQ ID NO: 36 SEQ ID NO: 37 SEQ ID NO: 38 T366WNO: 35 AEM AEM 2.1 SEQ ID SEQ ID NO: 40 SEQ ID NO: 41 SEQ ID NO: 42 2Y407T NO: 39 AEM 2.2 SEQ ID SEQ ID NO: 44 SEQ ID NO: 45 SEQ ID NO: 46T366Y NO: 43 AEM AEM 3.1 S364H/F405A SEQ ID SEQ ID NO: 48 SEQ ID NO: 49SEQ ID NO: 50 3 NO: 47 AEM 3.2 Y349T/T394F SEQ ID SEQ ID NO: 52 SEQ IDNO: 53 SEQ ID NO: 54 NO: 51 AEM AEM 4.1 K370D/K392D/ SEQ ID SEQ ID NO:56 SEQ ID NO: 57 SEQ ID NO: 58 4 K409D NO: 55 AEM 4.2 D356K*/E357K/ SEQID SEQ ID NO: 60 SEQ ID NO: 61 SEQ ID NO: 62 D399K NO: 59 DiS 1 DiS 1.1SEQ ID SEQ ID NO: 64 SEQ ID NO: 65 SEQ ID NO: 66 Y349C NO: 63 DiS 1.2SEQ ID SEQ ID NO: 68 SEQ ID NO: 69 SEQ ID NO: 70 S354C NO: 67 DiS 2 DiS2.1 SEQ ID — SEQ ID NO: 72 — C. term. KSCDKT NO: 71 DiS 2.2 SEQ ID — SEQID NO: 74 — C. term. GEC NO: 73 AEM AEM 1.1 + DiS 1.1 SEQ ID SEQ ID NO:76 SEQ ID NO: 77 SEQ ID NO: 78 1 + Y349C/T366S/L368A/ NO: 75 DiS 1 Y407VAEM 1.2 + DiS 1.2 SEQ ID SEQ ID NO: 80 SEQ ID NO: 81 SEQ ID NO: 82S354C/T366W NO: 79 AEM AEM 1.1 + DiS 2.1 SEQ ID — SEQ ID NO: 84 — 1 +T366S/L368A/Y407V/ NO: 83 DiS 2 C. term. KSCDKT AEM 1.2 + DiS 2.2 SEQ ID— SEQ ID NO: 86 — T366W/C term. GEC NO: 85 AEM AEM 1.1 + DiS 2.2 SEQ ID— SEQ ID NO: 88 — 1 + T366S/L368A/Y407V/ NO: 87 DiS C term. GEC 2Inv AEM2.1 + DiS 2.1 SEQ ID — SEQ ID NO: 90 — T366W/C. term. KSCDKT NO: 89 AEMAEM 3.1 +DiS 2.1 SEQ ID — SEQ ID NO: 92 — 3 + S364H/F405A/C term. NO: 91DiS 2 KSCDKT AEM 3.2 + DiS 2.2 SEQ ID — SEQ ID NO: 94 — Y349T/T394F/Cterm. GEC NO: 93 AEM AEM 4.1 + DiS 2.1 SEQ ID — SEQ ID NO: 96 — 4 +K370D/K392D/K409D/ NO: 95 DiS 2 C. term. KSCDKT AEM 4.2 + DiS 2.2 SEQ ID— SEQ ID NO: 98 — D356K*/E357K/D399K/ NO: 97 C term. GEC *With respectto the sequences that have an E at position 356 (e.g., SEQ ID NOs: 29and 30), this mutation is E356K.

Other CH3 AEMs that may be used in TFcAs include the following pairs ofaa modifications, wherein the substitution(s) to the first and thesecond member of a pair of AEM modifications are separated. by “and”:

1) F405A and T394F; S364D and Y349K; S364E and L368K; S364E Y349K; S364Fand K370G; S364H and Y349K; S364H and Y349T; S364Y and K370G; T411K andK370E; V397S/F405A and T394F; K370R/T411K and K370E/T411E; L351E/S364Dand Y349K/L351K; L351E/S364E and Y349K/L351K; L351E/T366D andL351K/T366K; P395T/V397S/F405A and T394F; S364D/K370G and S364Y/K370R;S364D/T394F and Y349K/F405 A; S364E/F405A and Y349K/T394F; S364E/F405Sand Y349/T394Y; S364E/T411E and Y349K/D401K; S364H/D401K andY349T/T411E; S364H/T394F and Y349T/F405A; Y349C/S364E and Y349K/S354C;L351E/S364D/F405A and Y349K/L351K/T394F; L351K/S364H/D401K andY349T/L351E/T411E; S364E/T411E/F405A and Y349K/T394F/D401K;S364H/D401K/F405A and Y349T/T394F/T411E; S364H/F405A/T411E andY349T/T394F/D401K (WO2011/028952).

2) T366W and Y407A; T366W and T366S; L368A and Y407Y; K409E and D399K;K409E and D399R; and K409D and D399K; K409D and D399R; K392E and D399R;K392E and D399K; K392D and D399R; and K392D and D399R (WO2009/089004).

3) 1366W and Y407A; F405A and T394W; Y407T and T366Y; T366Y/F405A andT394W/Y407T; T366W/F405W and T394S/Y407A; F405W/Y407A and T366W/T394S;and F405W and T394S (U.S. Pat. No. 7,642,228).

Generally, any other AEM or DiS described in the art may be used.

A CH3 domain may also comprise an aa sequence that differs from that ofa CH3 aa sequence provided herein, e.g., SEQ ID NOs:27-98, in at most,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 30 aas. In certainembodiments, a CH3 domain comprises an aa sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to a CH3 aa sequenceprovided herein, e.g., SEQ ID NOs:27-98. As several antibody effectorfunctions, e.g., ADCC and CDC are mediated at least in part throughareas in CH3 domains, aa changes that can be made to CH3 domains includechanges affecting the effector function(s) of Fc regions. Exemplarymodifications that may be made to CH3 domains are further describedherein.

Exemplary Fc Regions

An Fc region of a TFcA comprises one or more of a hinge, CH2 domain, CH3domain and CH4 domain, which may be full length or not and which may bewild type or with aa modifications. The Fc domains may be from humanimmunoglobulins (“Igs”) or non-human Igs, e.g., mouse Igs, and may befrom any type or isotype of an Ig, such as an IgG (e.g., IgG1, IgG2,IgG3 and IgG4) or IgA (e.g., IgA1 and IgA2).

In certain embodiments, a TFcA comprises a TFc that comprises a firstand/or a second Fc region from a human immunoglobulin, e.g., IgG1. An Fcregion preferably comprises in a contiguous amino to carboxyl terminalorder: an IgG1 hinge or portion thereof (e.g., a core and lower hinge),an IgG1 CH2 domain and an IgG1 CH3 domain. Fc regions may comprise anycombination of an IgG1 hinge (or portion thereof), IgG1 CH2 domain andIgG1 CH3 domains set forth herein, provided that the TFcA has thedesired activity and stability.

In certain embodiments, a TFc comprises a first and/or a second Fcregion that is a hybrid Fc region. A hybrid Fc region may comprise Fcdomains from two or more IgG subclasses IgG1, IgG2, IgG3 and IgG4. Inone embodiment, a hybrid Fc region comprises in a contiguous amino tocarboxyl terminal order: an IgG1 upper hinge, an IgG4 middle and lowerhinge, an IgG4 CH2 domain and an IgG1 CH3 domain Exemplary IgG1/IgG4hybrid Fc regions may comprise any combination of an IgG1 upper hinge,IgG4 core hinge, IgG4 lower hinge, IgG 4 CH2 domain and IgG1 CH3 domainsset forth herein, provided that the TFcA comprising the TFc has thedesired activity and stability.

In certain embodiments, an Fc region does not comprise a full lengthhinge. For example, a TFcBA may comprise a first Fc region comprising afull length hinge and a second Fc region comprising a hinge thatconsists of the core and/or lower hinge and does not comprise an upperhinge.

In certain embodiments, a TFc comprises a hinge that has been modifiedto comprise a cysteine for forming a disulfide bond with anothercysteine in the other Fc region, to thereby stabilize the TFc. In oneembodiment, an IgG1 hinge comprises the substitutions H224C and T225C(e.g., SEQ ID NO:18; FIG. 2). In another embodiment, a hinge comprisesthe substitution T223C (e.g., SEQ ID NO: 19; FIG. 2).

In one embodiment, a TFcA comprises an IgG1 TFc comprising in amino tocarboxyl terminal order: i) an IgG1 hinge selected from the group ofhinges consisting of the aa sequences set forth in SEQ ID NO:4 (fulllength IgG1 hinge), SEQ ID NO:18 (SEQ ID NO:4 with H224C/T225C), SEQ IDNO:19 (SEQ ID NO:4 with T223C) and SEQ ID NO:23 (middle and lower IgG1hinge only); ii) an IgG1 CH2 domain comprising SEQ ID NO:261 or 25; andiii) a CH3 domain comprising an aa sequence selected from the group ofCH3 domains consisting of an aa sequence set forth in SEQ ID NOs:31-98(FIG. 3). In another embodiment, a TFcA comprises an IgG1/IgG4 hybridTFc comprising in amino to carboxyl terminal order: i) a hinge selectedfrom the group of hinges consisting of an aa sequence set forth in SEQID NO:20 (IgG1 upper hinge and IgG4 core and lower hinge), SEQ ID NO:21(SEQ ID NO:20 with H224C/T225C), SEQ ID NO:22 (SEQ ID NO:20 with T223C)and SEQ ID NO:24 (middle and lower IgG4 hinge only); ii) an IgG4 CH2domain comprising SEQ ID NO:262 or 26; and iii) a CH3 domain comprisingan aa sequence selected from the group of CH3 domains consisting of theaa sequences SEQ ID NOs:31-98 (FIG. 3). Exemplary combinations of hingesand CH3 domains are set forth in Table 7, wherein a “yes’ indicates acombination that may be used, and wherein compatible modifications areseparated from others by a blank row.

TABLE 7 Exemplary combinations of hinges and CH3 domains for forming Fcregions Hinge IgG1 H224C/ IgG1 Partial Hybrid IgG1 T225C T223C IgG1H224C/ Hybrid Partial SEQ SEQ SEQ SEQ Hybrid T225C T223C IgG4 ID ID IDID SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 18 NO: 19 NO: 23 NO: 20 NO: 21NO: 22 NO: 24 CH3 AEM 1.1 yes yes yes yes yes yes yes yes SEQ ID NOs:31-34 AEM 1.2 yes yes yes yes yes yes yes yes SEQ ID NOs: 35-38 AEM 2.1yes yes yes yes yes yes yes yes SEQ ID NOs: 39-42 AEM 2.2 yes yes yesyes yes yes yes yes SEQ ID NOs: 43-46 AEM 3.1 yes yes yes yes yes yesyes yes SEQ ID NOs: 47-50 AEM 3.2 yes yes yes yes yes yes yes yes SEQ IDNOs: 51-54 AEM 4.1 yes yes yes yes yes yes yes yes SEQ ID NOs: 55-58 AEM4.2 yes yes yes yes yes yes yes yes SEQ ID NOs: 59-62 DiS 1.1 yes yesyes yes yes yes yes yes SEQ ID NOs: 63-66 DiS 1.2 yes yes yes yes yesyes yes yes SEQ ID NOs: 67-70 DiS 2.1 yes yes yes yes yes yes yes yesSEQ ID NOs: 71-72 DiS 2.2 yes yes yes yes yes yes yes yes SEQ ID NOs:73-74 AEM 1.1 yes yes yes yes yes yes yes yes DiS 1.1 SEQ ID NOs: 75-78AEM 1.2 yes yes yes yes yes yes yes yes DiS 1.2 SEQ ID NOs: 79-82 AEM1.1 yes yes yes yes yes yes yes yes DiS 2.1 SEQ ID NOs: 83-84 AEM 1.2yes yes yes yes yes yes yes yes DiS 2.2 SEQ ID NOs: 85-86 AEM 1.1 yesyes yes yes yes yes yes yes DiS 2.2 SEQ ID NOs: 87-88 AEM 1.2 yes yesyes yes yes yes yes yes DiS 2.1 SEQ ID NOs: 89-90 AEM 3.1 yes yes yesyes yes yes yes yes DiS 2.1 SEQ ID NOs: 91-92 AEM 3.2 yes yes yes yesyes yes yes yes DiS 2.2 SEQ ID NOs: 93-94 AEM 4.1 yes yes yes yes yesyes yes yes DiS 2.1 SEQ ID NOs: 95-96 AEM 4.2 yes yes yes yes yes yesyes yes DiS 2.2 SEQ ID NOs: 97-98

In one embodiment, a TFcA comprises a TFc that comprises an IgG1 Fcregion comprising a hinge comprising SEQ ID NO:4, a CH2 domaincomprising SEQ ID NO:25 and a CH3 domain comprising SEQ ID NO:29, andmay form, e.g., an IgG1 Fc region comprising SEQ ID NO:99 (see Table 8and FIG. 4). Other combinations of IgG1 hinges, IgG1 CH2 domain, andIgG1 CH3 domains and exemplary IgG1 Fcs created by such combinations areprovided in Table 8. The aa sequences of the exemplary IgG1 Fcs listedin Table 8 (SEQ ID NOs:99-132) are provided in FIG. 4. Compatible IgG1Fcs in Table 8 are separated from other IgG1 Fcs by a blank row.

TABLE 8 SEQ ID NOs of exemplary combinations of IgG1 hinges, CH2 domainand CH3 domain forming exemplary IgG1 Fcs IgG1 Hinge IgG1 CH2 IgG1 CH3IgG1 Fc SEQ ID NO: 4 SEQ ID NO: 25 SEQ ID NO: 29 SEQ ID NO: 99 SEQ IDNO: 23 SEQ ID NO: 25 SEQ ID NO: 29 SEQ ID NO: 100 SEQ ID NO: 4 SEQ IDNO: 25 SEQ ID NO: 33 SEQ ID NO: 101 SEQ ID NO: 23 SEQ ID NO: 25 SEQ IDNO: 37 SEQ ID NO: 102 SEQ ID NO: 4 SEQ ID NO: 25 SEQ ID NO: 37 SEQ IDNO: 103 SEQ ID NO: 23 SEQ ID NO: 25 SEQ ID NO: 33 SEQ ID NO: 104 SEQ IDNO: 4 SEQ ID NO: 25 SEQ ID NO: 77 SEQ ID NO: 105 SEQ ID NO: 23 SEQ IDNO: 25 SEQ ID NO: 81 SEQ ID NO: 106 SEQ ID NO: 4 SEQ ID NO: 25 SEQ IDNO: 81 SEQ ID NO: 107 SEQ ID NO: 23 SEQ ID NO: 25 SEQ ID NO: 77 SEQ IDNO: 108 SEQ ID NO: 4 SEQ ID NO: 25 SEQ ID NO: 41 SEQ ID NO: 109 SEQ IDNO: 23 SEQ ID NO: 25 SEQ ID NO: 45 SEQ ID NO: 110 SEQ ID NO: 4 SEQ IDNO: 25 SEQ ID NO: 45 SEQ ID NO: 111 SEQ ID NO: 23 SEQ ID NO: 25 SEQ IDNO: 41 SEQ ID NO: 112 SEQ ID NO: 18 SEQ ID NO: 25 SEQ ID NO: 33 SEQ IDNO: 113 SEQ ID NO: 4 SEQ ID NO: 25 SEQ ID NO: 77 SEQ ID NO: 114 SEQ IDNO: 19 SEQ ID NO: 25 SEQ ID NO: 33 SEQ ID NO: 115 SEQ ID NO: 4 SEQ IDNO: 25 SEQ ID NO: 77 SEQ ID NO: 116 SEQ ID NO: 4 SEQ ID NO: 25 SEQ IDNO: 84 SEQ ID NO: 117 SEQ ID NO: 23 SEQ ID NO: 25 SEQ ID NO: 86 SEQ IDNO: 118 SEQ ID NO: 4 SEQ ID NO: 25 SEQ ID NO: 86 SEQ ID NO: 119 SEQ IDNO: 23 SEQ ID NO: 25 SEQ ID NO: 84 SEQ ID NO: 120 SEQ ID NO: 4 SEQ IDNO: 25 SEQ ID NO: 88 SEQ ID NO: 121 SEQ ID NO: 23 SEQ ID NO: 25 SEQ IDNO: 90 SEQ ID NO: 122 SEQ ID NO: 4 SEQ ID NO: 25 SEQ ID NO: 90 SEQ IDNO: 123 SEQ ID NO: 23 SEQ ID NO: 25 SEQ ID NO: 88 SEQ ID NO: 124 SEQ IDNO: 4 SEQ ID NO: 25 SEQ ID NO: 92 SEQ ID NO: 125 SEQ ID NO: 23 SEQ IDNO: 25 SEQ ID NO: 94 SEQ ID NO: 126 SEQ ID NO: 4 SEQ ID NO: 25 SEQ IDNO: 94 SEQ ID NO: 127 SEQ ID NO: 23 SEQ ID NO: 25 SEQ ID NO: 92 SEQ IDNO: 128 SEQ ID NO: 4 SEQ ID NO: 25 SEQ ID NO: 96 SEQ ID NO: 129 SEQ IDNO: 23 SEQ ID NO: 25 SEQ ID NO: 98 SEQ ID NO: 130 SEQ ID NO: 4 SEQ IDNO: 25 SEQ ID NO: 98 SEQ ID NO: 131 SEQ ID NO: 23 SEQ ID NO: 25 SEQ IDNO: 96 SEQ ID NO: 132

In one embodiment, a TFcA comprises a TFc that comprises an IgG1/IgG4 Fcregion comprising a hinge comprising SEQ ID NO:20, a CH2 domaincomprising SEQ ID NO:26 and a CH3 domain comprising SEQ ID NO:29, andmay form, e.g., an IgG1/IgG4 hybrid Fc region comprising SEQ ID NO:133(see Table 9 and FIG. 4). Other combinations of IgG1/IgG4 hinges, IgG4CH2 domain, and IgG1 CH3 domains and exemplary IgG1/IgG4 hybrid Fcscreated by such combinations are provided in Table 9. The aa sequencesof the exemplary IgG1/IgG4 hybrid Fcs listed in Table 9 (SEQ IDNOs:133-166) are provided in FIG. 5. Compatible IgG1 Fcs in Table 9 areseparated from other IgG1 Fcs by a blank row.

TABLE 9 Exemplary combinations of IgG1/IgG4 hinges, CH2 domain and CH3domain forming exemplary IgG1/IgG4 hybrid Fcs Hinge IgG4 CH2 IgG1 CH3IgG1/IgG4 Fc SEQ ID NO: 20 SEQ ID NO: 26 SEQ ID NO: 29 SEQ ID NO: 133SEQ ID NO: 24 SEQ ID NO: 26 SEQ ID NO: 29 SEQ ID NO: 134 SEQ ID NO: 20SEQ ID NO: 26 SEQ ID NO: 33 SEQ ID NO: 135 SEQ ID NO: 24 SEQ ID NO: 26SEQ ID NO: 37 SEQ ID NO: 136 SEQ ID NO: 20 SEQ ID NO: 26 SEQ ID NO: 37SEQ ID NO: 137 SEQ ID NO: 24 SEQ ID NO: 26 SEQ ID NO: 33 SEQ ID NO: 138SEQ ID NO: 20 SEQ ID NO: 26 SEQ ID NO: 77 SEQ ID NO: 139 SEQ ID NO: 24SEQ ID NO: 26 SEQ ID NO: 81 SEQ ID NO: 140 SEQ ID NO: 20 SEQ ID NO: 26SEQ ID NO: 81 SEQ ID NO: 141 SEQ ID NO: 24 SEQ ID NO: 26 SEQ ID NO: 77SEQ ID NO: 142 SEQ ID NO: 20 SEQ ID NO: 26 SEQ ID NO: 41 SEQ ID NO: 143SEQ ID NO: 24 SEQ ID NO: 26 SEQ ID NO: 45 SEQ ID NO: 144 SEQ ID NO: 20SEQ ID NO: 26 SEQ ID NO: 45 SEQ ID NO: 145 SEQ ID NO: 24 SEQ ID NO: 26SEQ ID NO: 41 SEQ ID NO: 146 SEQ ID NO: 21 SEQ ID NO: 26 SEQ ID NO: 33SEQ ID NO: 147 SEQ ID NO: 24 SEQ ID NO: 26 SEQ ID NO: 77 SEQ ID NO: 148SEQ ID NO: 22 SEQ ID NO: 26 SEQ ID NO: 33 SEQ ID NO: 149 SEQ ID NO: 24SEQ ID NO: 26 SEQ ID NO: 77 SEQ ID NO: 150 SEQ ID NO: 20 SEQ ID NO: 26SEQ ID NO: 84 SEQ ID NO: 151 SEQ ID NO: 24 SEQ ID NO: 26 SEQ ID NO: 86SEQ ID NO: 152 SEQ ID NO: 20 SEQ ID NO: 26 SEQ ID NO: 86 SEQ ID NO: 153SEQ ID NO: 24 SEQ ID NO: 26 SEQ ID NO: 84 SEQ ID NO: 154 SEQ ID NO: 20SEQ ID NO: 26 SEQ ID NO: 88 SEQ ID NO: 155 SEQ ID NO: 24 SEQ ID NO: 26SEQ ID NO: 90 SEQ ID NO: 156 SEQ ID NO: 20 SEQ ID NO: 26 SEQ ID NO: 90SEQ ID NO: 157 SEQ ID NO: 24 SEQ ID NO: 26 SEQ ID NO: 88 SEQ ID NO: 158SEQ ID NO: 20 SEQ ID NO: 26 SEQ ID NO: 92 SEQ ID NO: 159 SEQ ID NO: 24SEQ ID NO: 26 SEQ ID NO: 94 SEQ ID NO: 160 SEQ ID NO: 20 SEQ ID NO: 26SEQ ID NO: 94 SEQ ID NO: 161 SEQ ID NO: 24 SEQ ID NO: 26 SEQ ID NO: 92SEQ ID NO: 162 SEQ ID NO: 20 SEQ ID NO: 26 SEQ ID NO: 96 SEQ ID NO: 163SEQ ID NO: 24 SEQ ID NO: 26 SEQ ID NO: 98 SEQ ID NO: 164 SEQ ID NO: 20SEQ ID NO: 26 SEQ ID NO: 98 SEQ ID NO: 165 SEQ ID NO: 24 SEQ ID NO: 26SEQ ID NO: 96 SEQ ID NO: 166

Fc regions for use in TFcAs may also comprise aa sequences that differfrom those described herein, e.g., SEQ ID NOs:99-166, in one or more aamodifications, e.g., aa deletions, additions or substitutions. Incertain embodiments, an Fc region comprises an aa sequence that differsfrom a sequence set forth herein, e.g., from a sequence selected fromthe group consisting of SEQ ID NOs:99-166, in at most, 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50 aas. In certainembodiments, an Fc region comprises an aa sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to that of a sequenceset forth herein, e.g., a sequence selected from the group consisting ofSEQ ID NOs:99-166. For example, the CH3 domain of an Fc regionconsisting of an aa sequence selected from the group consisting of SEQID NOs:99-166 may comprise a deletion of the C-terminal lysine and/orE356D and/or M358L. As several antibody effector functions, e.g., ADCCand CDC, are mediated through Fc regions, aa changes that can be made toFc regions include changes affecting the effector function(s) of Fcregions. Exemplary mutations to these domains are set forth herein. Anyof these aa modifications are permitted provided that the Fc regionretains the desired properties, e.g., biological activity, stability andlow immunogenicity.

Exemplary Fc Modifications Affecting Effector Activity

TFcAs may comprise TFcs comprising aa modifications affecting theeffector activity of Fc regions. Exemplary aa modifications are setforth below.

Replacements of aa residues in the Fc portion to alter antibody effectorfunction are known in the art (see, e.g., U.S. Pat. Nos. 5,648,260 and5,624,821). The Fc portion of an antibody mediates several importanteffector functions e.g., cytokine induction, antibody-dependent cellularcytotoxicity (“ADCC”), phagocytosis, complement dependent cytotoxicity(CDC) and half-life/clearance rate of antibody and antigen-antibodycomplexes. In some cases these effector functions are desirable for atherapeutic antibody but in other cases might be unnecessary or evendeleterious, depending on the therapeutic objectives. Certain human IgGisotypes, particularly IgG1 and IgG3, mediate ADCC and CDC via bindingto FcγRs and complement CIq, respectively. Neonatal Fc receptors (FcRn)are the critical components determining the circulating half-life ofantibodies.

In one embodiment, a TFcA retains one or more of, and preferably all ofthe following attributes: ADCC and antibody-dependent cellularphagocytosis (ADCP) that in humans are determined by interactions withactivating FcγRI, FcγRIIa/c, FcγRIIIa and inhibitory FcγRIIb receptors;CDC that is triggered by antibody binding to the components of thecomplement system; and long half-life that is mediated via activerecycling by the neonatal Fc receptor (FcRn). When desired, all of thesefunctions can be tuned to optimize the effectiveness of an anti-cancertherapy.

Certain aa modifications, e.g., the addition, deletion and/orsubstitution of one or more aas may be made to an immunoglobulinconstant region to reduce or increase the natural biological activitiesof the constant domains, such as those set forth above.

In certain embodiments, a TFcA (e.g., a TFcBA) comprises an aamodification (e.g., an aa substitution, addition or deletion) in an Fcregion that alters one or more antigen-independent effector functions ofthe domain, e.g., the circulating half-life of a protein comprising thedomain Exemplary antibodies exhibit either increased or decreasedbinding to FcRn when compared to antibodies lacking such aa changes and,therefore, have an increased or decreased half-life in serum,respectively. Antibodies comprising Fc variants with improved affinityfor FcRn are anticipated to have longer serum half-lives, whereas thosecomprising Fc variants with decreased FcRn binding affinity are expectedto have shorter half-lives. In one embodiment, a TFcA with altered FcRnbinding comprises at least one Fc region having one or more aa changeswithin the “FcRn binding loop” of an Fc region. The FcRn binding loop iscomprised of aa residues 280-299 (EU) of a wild type, full length, Fc.In certain embodiments, a TFcA having altered FcRn binding affinitycomprises at least one Fc region having one or more aa substitutionswithin the 15 A FcRn “contact zone.” The term 15 A FcRn “contact zone”includes residues at the following positions of a wild type, full-lengthFc domain: 243-261, 275-280, 282-293, 302-319, 336-348, 367, 369,372-389, 391, 393, 408, 424-440 (EU). In certain embodiments, a TFcAhaving altered FcRn binding affinity comprises at least one Fc region(e.g., one or two Fc moieties) having one or more aa changes at an aaposition corresponding to any one of the following EU positions: 256,277-281, 283-288, 303-309, 313, 338, 342, 376, 381, 384, 385, 387, 434(e.g., N434A or N434K), and 438. Exemplary aa changes that alter FcRnbinding activity are disclosed in International PCT Publication No.WO05/047327.

Additional Fc modifications that enhance FcRn binding includesubstitutions at positions 259, 308, 428, and 434, e.g., 259I, 308F,428L, 428M, 434S, 434H, 434F, 434Y, 434M, 428L/434S, 259I/308F and259I/308F/428L. Other variants that increase Fc binding to FcRn include:250E, 250Q, 428L, 428F, 250Q/428L (Hinton et al., 2004, J. Biol. Chem.279(8): 6213-6216, Hinton et al. 2006 Journal of Immunology176:346-356), 256A, 272A, 286A, 305A, 307A, 307Q, 31 1A, 312A, 376A,378Q, 380A, 382A, 434A (Shields et al, Journal of Biological Chemistry,2001, 276(9):6591-6604), 252F, 252T, 252Y, 252W, 254T, 256S, 256R, 256Q,256E, 256D, 256T, 309P, 311S, 433R, 433S, 4331, 433P, 433Q, 434H, 434F,434Y, 252Y/254T/256E, 433K/434F/436H, 308T/309P/311S (Dall Acqua et al.Journal of Immunology, 2002, 169:5171-5180, Dall'Acqua et al., 2006,Journal of Biological Chemistry 281:23514-23524). Other modificationsfor modulating FcRn binding are described in Yeung et al., 2010, JImmunol, 182:7663-7671.

In some embodiments, a TFcA comprises an Fc variant comprising an aachange that alters the antigen-dependent effector functions of thepolypeptide, in particular ADCC or complement activation, e.g., ascompared to a wild type Fc region. In exemplary embodiment, saidantibodies exhibit altered binding to an Fc gamma receptor (e.g., CD16).Such antibodies exhibit either increased or decreased binding to FcγRswhen compared to wild type polypeptides and, therefore, mediate enhancedor reduced effector function, respectively. Fc variants with improvedaffinity for FcγRs are anticipated to enhance effector function, andsuch proteins may have useful applications in methods of treatingmammals where target molecule destruction is desired, e.g., in tumortherapy. In contrast, Fc variants with decreased FcγR binding affinityare expected to reduce effector function. In one embodiment, a TFcAcomprises at least one altered antigen-dependent effector functionselected from the group consisting of opsonization, phagocytosis,complement dependent cytotoxicity, antigen-dependent cellularcytotoxicity (ADCC), or effector cell modulation as compared to a TFcAcomprising a wild type Fc region.

In certain embodiments, a TFcA exhibits altered binding to an activatingFcγR (e.g. FcγRI, FcγRIIa, or FcγRIIIa). In certain embodiments, a TFcAexhibits altered binding affinity to an inhibitory FcγR (e.g. FcγRIIb).In other embodiments, a TFcA having increased FcγR binding affinity(e.g. increased FcγRIIIa binding affinity) comprises at least one Fcdomain having an aa change at an aa position corresponding to one ormore of the following positions: 239, 268, 298, 332, 334, and 378 (EU).In certain embodiments, a TFcA having decreased FcγR binding affinity(e.g. decreased FcγRI, FcγRII, or FcγRIIIa binding affinity) comprisesat least one Fc domain having an aa substitution at an aa positioncorresponding to one or more of the following positions: 234, 236, 239,241, 251, 252, 261, 265, 268, 293, 294, 296, 298, 299, 301, 326, 328,332, 334, 338, 376, 378, and 435 (EU).

In certain embodiments, a TFcA having increased complement bindingaffinity (e.g. increased C1q binding affinity) comprises an Fc domainhaving an aa change at an aa position corresponding to one or more ofthe following positions: 251, 334, 378, and 435 (EU). In certainembodiments, a TFcA having decreased complement binding affinity (e.g.decreased C1q binding affinity) comprises an Fc domain having an aasubstitution at an aa position corresponding to one or more of thefollowing positions: 239, 294, 296, 301, 328, 333, and 376 (EU).Exemplary aa changes that alter FcγR or complement binding activity aredisclosed in International PCT Publication No. WO05/063815. In certainembodiments, a TFcA may comprise one or more of the following specificFc region substitutions: S239D, S239E, M252T, H268D, H268E, I332D,I332E, N434A, and N434K (EU).

Other Fc variants that reduce binding to FcγRs and/or complement includevariants comprising one or more of the following aa substitutions: 34G,235G, 236R, 237K, 267R, 269R, 325L, 328R, 236R/328R, 297A, 234A, 235A,237A, 318A, 228P, 236E, 268Q, 309L, 330S, 331 S, 220S, 226S, 229S, 238S,233P, and 234V. Removal of the glycosylation at position 297 (see below)also reduces binding to FcγRs.

Fc modifications that improve binding to FcγRs and/or complement includevariants comprising one or more of the following aa substitutions: 236A,239D, 239E, 268D, 267E, 268E, 268F, 324T, 332D, and 332E. Preferredvariants include but are not limited to 239D/332E, 236A/332E,236A/239D/332E, 268F/324T, 267E/268F, 267E/324T, and 267E/268F/324T.Other modifications for enhancing FcγR and complement interactionsinclude but are not limited to substitutions 298A, 333A, 334A, 326A,2471, 339D, 339Q, 280H, 290S, 298D, 298V, 243L, 292P, 300L, 396L, 3051,and 396L.

Variants that improve binding to FcγRllb include variants comprising oneor more of the following aa substitutions: 234D, 234E, 234W, 235D, 235F,235R, 235Y, 236D, 236N, 237D, 237N, 239D, 239E, 266M, 267D, 267E, 268D,268E, 327D, 327E, 328F, 328W, 328Y and 332E, 235Y/267E, 236D/267E,239D/268D, 239D/267E, 267E/268D, 267E/268E, and 267E/328F.

Fc modifications modulating Fc are described in Strohl, 2009, CurrentOpinion in Biotechnology 20:685-691.

A TFcA may also comprise an aa substitution that alters theglycosylation of the TFcA. For example, an immunoglobulin constantregion of a TFcA may comprise an Fc domain having a mutation leading toreduced glycosylation (e.g., N- or O-linked glycosylation) or maycomprise an altered glycoform of the wild type Fc domain (e.g., a lowfucose or fucose-free glycan). An “engineered glycoform” refers to acarbohydrate composition that is covalently attached to an Fc region,wherein said carbohydrate composition differs chemically from that of aparent Fc region. Engineered glycoforms may be useful for a variety ofpurposes, including but not limited to enhancing or reducing effectorfunction. Engineered glycoforms may be generated by a variety of methodsknown in the art (U.S. Pat. No. 6,602,684; US Pat Pub No. 2010-0255013;US Pat Pub No. 2003-0003097; WO 00/61739A1; WO 01/29246A1; WO02/31140A1; WO 02/30954A1); (Potelligent technology (Biowa, Inc.,Princeton, N.J.); and GlycoMAb glycosylation engineering technology(Glycart Biotechnology AG, Zurich, Switzerland). Many of thesetechniques are based on controlling the level of fucosylated and/orbisecting oligosaccharides that are covalently attached to the Fcregion, for example by expressing an Fc polypeptide in various organismsor cell lines, engineered or otherwise (for example Lec-13 CHO cells orrat hybridoma YB2/0 cells), by regulating enzymes involved in theglycosylation pathway (for example FUT8 [a1, 6-fucosyltranserase] and/or(31-4-N-acetylglucosaminyl, transferase III [GnTIll]), or by modifyingcarbohydrate(s) after the Fc polypeptide has been expressed.

In exemplary embodiments, an aa change, e.g., an aa substitution,results in an Fc region comprising reduced glycosylation of the N-linkedglycan normally found at aa position 297 (EU). The Fc region may alsocomprise a low fucose or fucose free glycan at aa position 297 (EU). Incertain embodiments, the TFcA has an aa substitution near or within aglycosylation motif, for example, an N-linked glycosylation motif thatcontains the aa sequence NXT or NXS. In a particular embodiment, a TFcAcomprises an aa substitution at an aa position corresponding to 297 or299 of Fc (EU) as further described herein. Exemplary aa substitutionsthat reduce or alter glycosylation are disclosed in International PCTPublication No. WO05/018572 and US Pat Pub No. 2007/0111281.

In other embodiments, a TFcA comprises at least one Fc domain having oneor more engineered cysteine residues or analog thereof that are locatedat the solvent-exposed surface. Preferably the engineered cysteineresidue or analog thereof does not interfere with the desired biologicalactivity of the TFcA. For example, it may be desirable that thealteration does not interfere with the ability of the Fc to bind to Fcreceptors (e.g. FcγRI, FcγRII, or FcγRIII) or complement proteins (e.g.C1q), or to trigger immune effector function (e.g., antibody-dependentcytotoxicity (ADCC), phagocytosis, or CDC). In certain embodiments,TFcAs comprise an Fc domain comprising at least one engineered freecysteine residue or analog thereof that is substantially free ofdisulfide bonding with a second cysteine residue. TFcAs may comprise anFc region having engineered cysteine residues or analogs thereof at oneor more of the following positions in the CH3 domain: 349-371, 390, 392,394-423, 441-446, and 446b (EU), and more specifically positions 350,355, 359, 360, 361, 389, 413, 415, 418, 422, 441, 443, and EU position446b.

Desired effector functions may also be obtained by choosing an Fc from aparticular immunoglobulin class or subclass, or by combining particularregions from particular immuoglobulin classes or subclasses, e.g., IgG1,IgG2, etc. For example, since ADCC and CDC (through binding of IgG tothe FcγRs and C1q, respectively) is mediated by residues located in thehinge and CH2 domain, and since IgG4 essentially lacks effectorfunctions, an Fc constructed by combining the hinge and CH2 domain ofIgG4 and the CH3 domain of IgG1 has much reduced effector functions. AnIgG1/IgG3 hybrid variant may be constructed by substituting IgG1positions in the CH2 and/or CH3 region with the amino acids from IgG3 atpositions where the two isotypes differ. Thus a hybrid variant IgGantibody may be constructed that comprises one or more of the followingsubstitutions: 274Q, 276K, 300F, 339T, 356E, 358M, 384S, 392N, 397M,4221, 435R, and 436F. In certain embodiments, an IgG1/IgG2 hybridvariant may be constructed by substituting IgG2 positions in the CH2and/or CH3 region with amino acids from IgG1 at positions where the twoisotypes differ. Thus a hybrid variant IgG antibody may be constructedthat comprises one or more of the following amino acid substations:233E, 234L, 235L, −236G (referring to an insertion of a glycine atposition 236), and 327A.

Exemplary TFc Linkers

A TFcA may comprise a TFc comprising a first Fc region that is linked toa second Fc region through a TFc linker A wide variety of linkers may beused provided that they are sufficiently flexible to allow properfolding of the TFc and of a TFcA comprising the TFc. In certainembodiments, a linker is biologically inert, e.g., mostly incapable ofinducing a biological response, e.g., an immune response.

A TFc linker may be 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70,70-80, 80-90 or at least 90-100 aas long. The size of a TFc linker maydepend on whether the second Fc region comprises a hinge, a portionthereof or no hinge at all. For example, when the second Fc regioncomprises a hinge, a shorter TFc linker may be used than when the secondFc region does not comprise a hinge. For example, when a second Fcregion does not comprise a hinge, a TFc linker may be longer by a numberof aas corresponding to the length of a hinge. When a second Fc regiondoes not comprise an upper hinge, a TFc linker may be longer by a numberof aas corresponding to the length of the upper hinge. In a preferredembodiment, a TFcA, e.g., a TFcA that comprises a second hingeconsisting of a middle and lower hinge, comprises a TFc linkercomprising from 35 to 45 aas, such as from 37 to 43 aas, such as from 38to 42 aas, such as from 39 to 41 aas, and more particularly, 40 aas.

A TFc linker may comprise a Gly-Ser linker A “Gly-Ser linker” refers toa peptide that consists of glycine and serine residues. An exemplaryGly-Ser linker comprises an aa sequence having the formula(Gly₄Ser)_(n), wherein n is a positive integer (e.g., 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20). For example, incertain embodiments, a TFc linker comprises or consists of (Gly₄Ser)₃ or(Gly₄Ser)₄ or (Gly₄Ser)₅ or (Gly₄Ser)₆ or (Gly₄Ser)₇ or (Gly₄Ser)₈. In apreferred embodiment, the TFc linker is (Gly₄Ser)₈.

Other linkers that may be used include those that comprise Gly and Ser,but not in a (G4S)n structure. For example, linkers may comprise(Gly-Gly-Ser)n or (Gly-Ser-Gly-Ser)n, wherein n is 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15 or more. Other linkers may comprise Pro orThr. Suitable linkers may be found in the Registry of StandardBiological Parts at http://partsregistry.org/Protein_domains/Linker (seealso, e.g., Crasto C J and Feng J A. LINKER: a program to generatelinker sequences for fusion proteins. Protein Eng 2000 May; 13(5) 309-12and George R A and Heringa J. An analysis of protein domain linkers:their classification and role in protein folding. Protein Eng 2002November; 15(11) 871-9).

In certain embodiments, a TFc linker comprises the following aasequence:

(SEQ ID NO: 169) TRPAPPSTATTAGSTPQPESASPSGKEPAASSPSSTNTGS

TFc linkers comprising an aa sequence that differs from SEQ ID NO:169 orfrom a (G4S)n sequence in at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 aasmay also be used.

A TFc linker may also be a non-peptide linker, such as a non-peptidepolymer. The term “non-peptide polymer,” refers to a biocompatiblepolymer including two or more repeating units linked to each other by acovalent bond excluding the peptide bond. Examples of the non-peptidepolymer include poly (ethylene glycol), poly (propylene glycol),copolymers of ethylene glycol and propylene glycol, polyoxyethylatedpolyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ether,biodegradable polymers such as PLA (poly (lactic acid) and PLGA (poly(lactic-glycolic acid), lipid polymers, chitins, and hyaluronic acid.The most preferred is poly (ethylene glycol) (PEG).

Exemplary TFcs

TFcAs may comprise a TFc comprising a first Fc region that is linked toa second Fc region through a TFc linker. In certain embodiments, a TFccomprises a first and a second Fc region that are identical to eachother. In other embodiments, the first and the second Fc regions differfrom each other in at least one aa (“heteromeric TFc”). A first and asecond Fc region may be any Fc region disclosed herein or a variationthereof. For example, a TFc may comprise a first Fc region thatcomprises a full length hinge, e.g., a full length IgG1 or IgG1/IgG4hybrid hinge, and a second Fc region that comprises a partial hinge,e.g., a hinge that is devoid of the upper hinge.

First and second Fc regions may be combined with any TFc linkerdescribed herein. As set forth above, generally the length of the TFclinker may depend on whether the second Fc region comprises a hinge, aportion thereof, or no hinge at all.

In certain embodiments, an IgG1 TFc comprises a first Fc regioncomprising SEQ ID NO:99 and a second Fc region comprising SEQ ID NO:100.Combinations of first and second Fc regions that may be used in IgG1TFcs are set forth in Table 10.

TABLE 10 Exemplary combinations of first and second Fc regions (shown inFIG. 4) for use in IgG1 TFcs First Fc region Second Fc region SEQ ID NO:99 SEQ ID NO: 100 SEQ ID NO: 101 SEQ ID NO: 102 SEQ ID NO: 103 SEQ IDNO: 104 SEQ ID NO: 105 SEQ ID NO: 106 SEQ ID NO: 107 SEQ ID NO: 108 SEQID NO: 109 SEQ ID NO: 110 SEQ ID NO: 111 SEQ ID NO: 112 SEQ ID NO: 113SEQ ID NO: 114 SEQ ID NO: 115 SEQ ID NO: 116 SEQ ID NO: 117 SEQ ID NO:118 SEQ ID NO: 119 SEQ ID NO: 120 SEQ ID NO: 121 SEQ ID NO: 122 SEQ IDNO: 123 SEQ ID NO: 124 SEQ ID NO: 125 SEQ ID NO: 126 SEQ ID NO: 127 SEQID NO: 128 SEQ ID NO: 129 SEQ ID NO: 130 SEQ ID NO: 131 SEQ ID NO: 132

In certain embodiments, an IgG1/IgG4 hybrid TFc comprises a first Fcregion comprising SEQ ID NO:133 and a second Fc region comprising SEQ IDNO:134. Combinations of first and second Fc regions that may be used inIgG1/IgG4 hybrid TFcs are set forth in Table 11.

TABLE 11 Exemplary combinations of first and second Fc regions (shown inFIG. 5) for use in IgG1/IgG4 TFcs First Fc region Second Fc region SEQID NO: 133 SEQ ID NO: 134 SEQ ID NO: 135 SEQ ID NO: 136 SEQ ID NO: 137SEQ ID NO: 138 SEQ ID NO: 139 SEQ ID NO: 140 SEQ ID NO: 141 SEQ ID NO:142 SEQ ID NO: 143 SEQ ID NO: 144 SEQ ID NO: 145 SEQ ID NO: 146 SEQ IDNO: 147 SEQ ID NO: 148 SEQ ID NO: 149 SEQ ID NO: 150 SEQ ID NO: 151 SEQID NO: 152 SEQ ID NO: 153 SEQ ID NO: 154 SEQ ID NO: 155 SEQ ID NO: 156SEQ ID NO: 157 SEQ ID NO: 158 SEQ ID NO: 159 SEQ ID NO: 160 SEQ ID NO:161 SEQ ID NO: 162 SEQ ID NO: 163 SEQ ID NO: 164 SEQ ID NO: 165 SEQ IDNO: 166

A TFc may comprise a combination of two Fcs set forth in Table 10 or 11,which are linked together through a TFc linker to form a contiguouspolypeptide comprising in amino to carboxyl terminal order: a first Fcregion, which is linked at its C-terminus to the N-terminus of a TFclinker, which is linked at its C-terminus to the N-terminus of thesecond Fc region. The TFc linker may comprise or consist of 20 to 50amino acids in length.

Exemplary TFcs may comprise: i) a first Fc region comprising a hingecomprising SEQ ID NO:4, a CH2 domain comprising SEQ ID NO:25, a CH3domain comprising SEQ ID NO:33; ii) a TFc linker comprising (G₄5)₈; andiii) a second Fc region comprising a hinge comprising SEQ ID NO:23, aCH2 domain comprising SEQ ID NO:25 and a CH3 domain comprising SEQ IDNO:37. An exemplary IgG1 TFc comprising this set of elements is a TFccomprising SEQ ID NO:171. Additional combinations of domains or elementsforming IgG1 and IgG1/IgG4 hybrid TFcs are provided in Table 12 and 13,respectively. Each of the elements or domains in Tables 12 and 13 isreferred to by its SEQ ID NO and the specific AEM and/or DiS that itcomprises. Each of the domains or elements in Tables 12 and 13 may belinked directly or indirectly.

The aa sequence of each of the TFcs listed in Table 12 (SEQ ID NOs:171,173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193 and 195) isprovided in FIG. 6. The aa sequence of each of the TFcs listed in Table13 (SEQ ID NOs:197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217,219 and 221) is provided in FIG. 7. The first column of Tables 12 and 13lists the name and SEQ ID NO of an exemplary TFc comprising the elementslisted in the corresponding row of the Table.

TABLE 12 IgG1 TFcs set forth in FIG. 6 IgG1 TFc First Fc TFc Second FcHinge CH2 CH3 linker Hinge CH2 CH3 23 SEQ ID SEQ ID SEQ ID NO: 33 (G₄S)₈SEQ ID SEQ ID SEQ ID NO: 37 SEQ ID NO: 4 NO: 25 T366S/L368A/ NO: 23 NO:25 T366W NO: 171 wild type Y407V partial 23A SEQ ID SEQ ID SEQ ID NO: 77(G₄S)₈ SEQ ID SEQ ID SEQ ID NO: 81 SEQ ID NO: 4 NO: 25 Y349C/T366S/ NO:23 NO: 25 S354C/T366W NO: 173 wild type L368A/Y407V partial 23B SEQ IDSEQ ID SEQ ID NO: 41 (G₄S)₈ SEQ ID SEQ ID SEQ ID NO: 45 SEQ ID NO: 4 NO:25 Y407T NO: 23 NO: 25 T366Y NO: 175 wild type partial 23C SEQ ID SEQ IDSEQ ID NO: 33 (G₄S)₈ SEQ ID SEQ ID SEQ ID NO: 37 SEQ ID NO: 18 NO: 25T366S/L368A/ NO: 23 NO: 25 T366W NO: 177 H224C/ Y407V partial T225C 23DSEQ ID SEQ ID SEQ ID NO: 33 (G₄S)₈ SEQ ID SEQ ID SEQ ID NO: 37 SEQ IDNO: 19 NO: 25 T366S/L368A/ NO: 23 NO: 25 T366W NO: 179 T223C Y407Vpartial 23E SEQ ID SEQ ID SEQ ID NO: 84 (G₄S)₈ SEQ ID SEQ ID SEQ ID NO:86 SEQ ID NO: 4 NO: 25 T366S/L368A/ NO: 23 NO: 25 T366W/C-term NO: 181wild type Y407V/C-term partial Cysteine Cysteine 23G SEQ ID SEQ ID SEQID NO: 33 (G₄S)₄ SEQ ID SEQ ID SEQ ID NO: 37 SEQ ID NO: 4 NO: 25T366S/L368A/ NO: 23 NO: 25 T366W NO: 183 wild type Y407V partial 23E SEQID SEQ ID SEQ ID NO: 84 (G₄S)₇ SEQ ID SEQ ID SEQ ID NO: 86 (35L) NO: 4NO: 25 T366S/L368A/ NO: 23 NO: 25 T366W/C-term SEQ ID wild typeY407V/C-term partial Cysteine NO: 185 Cysteine 23E SEQ ID SEQ ID SEQ IDNO: 88 (G₄S)₇ SEQ ID SEQ ID SEQ ID NO: 90 (35L) Inv NO: 4 NO: 25T366S/L368A/ NO: 23 NO: 25 T366W/C-term SEQ ID wild type Y407V/C-termpartial Cysteine Inv NO: 187 Cysteine Inv 23E SEQ ID SEQ ID SEQ ID NO:84 (G₄S)₆ SEQ ID SEQ ID SEQ ID NO: 86 (30L) NO: 4 NO: 25 T366S/L368A/NO: 23 NO: 25 T366W/C-term SEQ ID wild type Y407V/C-term partialCysteine NO: 189 Cysteine 23E SEQ ID SEQ ID SEQ ID NO: 84 (G₄S)₅ SEQ IDSEQ ID SEQ ID NO: 86 (25L) NO: 4 NO: 25 T366S/L368A/ NO: 23 NO: 25T366W/C-term SEQ ID wild type Y407V/C-term partial Cysteine NO: 191Cysteine 23I SEQ ID SEQ ID SEQ ID NO: 92 (G₄S)₈ SEQ ID SEQ ID SEQ ID NO:94 SEQ ID NO: 4 NO: 25 S364H/F405A NO: 23 NO: 25 Y349T/T394F NO: 193wild type partial 23J SEQ ID SEQ ID SEQ ID NO: 96 (G₄S)₈ SEQ ID SEQ IDSEQ ID NO: 98 SEQ ID NO: 4 NO: 25 K370D/K392D/ NO: 23 NO: 25D356K/E357K/D399K NO: 195 wild type K409D partial

TABLE 13 IgG1/IgG4 hybrid TFcs set forth in FIG. 7 IgG1/IgG4 hybrid TFcFirst Fc TFc Second Fc Hinge CH2 CH3 linker Hinge CH2 CH3 39 SEQ ID SEQID SEQ ID NO: 33 (G₄S)₈ SEQ ID SEQ ID SEQ ID NO: 37 SEQ ID NO: 20 NO: 26T366S/L368A/ NO: 24 NO: 26 T366W NO: 197 wild type Y407V partial 39A SEQID SEQ ID SEQ ID NO: 77 (G₄S)₈ SEQ ID SEQ ID SEQ ID NO: 81 SEQ ID NO: 20NO: 26 Y349C/T366S/ NO: 24 NO: 26 S354C/T366W NO: 199 wild typeL368A/Y407V partial 39B SEQ ID SEQ ID SEQ ID NO: 41 (G₄S)₈ SEQ ID SEQ IDSEQ ID NO: 45 SEQ ID NO: 20 NO: 26 Y407T NO: 24 NO: 26 T366Y NO: 201wild type partial 39C SEQ ID SEQ ID SEQ ID NO: 33 (G₄S)₈ SEQ ID SEQ IDSEQ ID NO: 37 SEQ ID NO: 21 NO: 26 T366S/L368A/ NO: 24 NO: 26 T366W NO:203 H224C/ Y407V partial T225C 23D SEQ ID SEQ ID SEQ ID NO: 33 (G₄S)₈SEQ ID SEQ ID SEQ ID NO: 37 SEQ ID NO: 22 NO: 26 T366S/L368A/ NO: 24 NO:26 T366W NO: 205 T223C Y407V partial 39E SEQ ID SEQ ID SEQ ID NO: 84(G₄S)₈ SEQ ID SEQ ID SEQ ID NO: 86 SEQ ID NO: 20 NO: 26 T366S/L368A/ NO:24 NO: 26 T366W/C-term NO: 207 wild type Y407V/C-term partial CysteineCysteine 39G SEQ ID SEQ ID SEQ ID NO: 33 (G₄S)₄ SEQ ID SEQ ID SEQ ID NO:37 SEQ ID NO: 20 NO: 26 T366S/L368A/ NO: 24 NO: 26 T366W NO: 209 wildtype Y407V partial 39E SEQ ID SEQ ID SEQ ID NO: 84 (G₄S)₇ SEQ ID SEQ IDSEQ ID NO: 86 (35L) NO: 20 NO: 26 T366S/L368A/ NO: 24 NO: 26T366W/C-term SEQ ID wild type Y407V/C-term partial Cysteine NO: 211Cysteine 39E SEQ ID SEQ ID SEQ ID NO: 88 (G₄S)₇ SEQ ID SEQ ID SEQ ID NO:90 (35L) Inv NO: 20 NO: 26 T366S/L368A/ NO: 24 NO: 26 T366W/C-term SEQID wild type Y407V/C-term partial Cysteine Inv NO: 213 Cysteine Inv 39ESEQ ID SEQ ID SEQ ID NO: 84 (G₄S)₆ SEQ ID SEQ ID SEQ ID NO: 86 (30L) NO:20 NO: 26 T366S/L368A/ NO: 24 NO: 26 T366W/C-term SEQ ID wild typeY407V/C-term partial Cysteine NO: 215 Cysteine 39E SEQ ID SEQ ID SEQ IDNO: 84 (G₄S)₅ SEQ ID SEQ ID SEQ ID NO: 86 (25L) NO: 20 NO: 26T366S/L368A/ NO: 24 NO: 26 T366W/C-term SEQ ID wild type Y407V/C-termpartial Cysteine NO: 217 Cysteine 39I SEQ ID SEQ ID SEQ ID NO: 92 (G₄S)₈SEQ ID SEQ ID SEQ ID NO: 94 SEQ ID NO: 20 NO: 26 S364H/F405A NO: 24 NO:26 Y349T/T394F NO: 219 wild type partial 39J SEQ ID SEQ ID SEQ ID NO: 96(G₄S)₈ SEQ ID SEQ ID SEQ ID NO: 98 SEQ ID NO: 20 NO: 26 K370D/K392D/ NO:24 NO: 26 D356K/E357K/D399K NO: 221 wild type K409D partial

In certain embodiments, a TFc comprises an aa sequence that differs fromthat of a TFc described herein, e.g., an aa sequence selected from thegroup consisting of SEQ ID NOs:171, 173, 175, 177, 179, 181, 183, 185,187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213,215, 217, 219 and 221, in at most 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 150, 200, 250 or 300 aas, provided that the TFc has the desiredbiological activity, such as effector function or lack thereof, properfolding, sufficient stability and solubility. Differences may be one ormore aa insertions, deletions and/or substitutions. In certainembodiments, a TFc comprises an aa sequence that is at least about 70%,75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to that of a TFcdescribed herein, e.g., an aa sequence selected from the groupconsisting of SEQ ID NOs:171, 173, 175, 177, 179, 181, 183, 185, 187,189, 191,193, 195, 197, 199, 201,203, 205, 207, 209, 211,213, 215, 217,219 and 221, provided that the TFcA comprising the TFc has the desiredbiological activity, such as effector function or lack thereof, properfolding, sufficient stability and/or sufficient solubility.

Exemplary Binding Sites

A TFcA may be a monovalent TFcA that comprises a single binding site.The single binding site may be located a the amino terminus or carboxylterminus of the TFc. The single binding site may be a Fab or an scFv.When the single binding site is a Fab, the monovalent TFcA comprises aheavy chain comprising the VH domain and optionally a CH1 domain and alight chain comprising the VL domain and optionally a CL domain.

A TFcA may also be a TFcBA comprising two binding sites, e.g., whereineach binding site binds to the same or to a different epitope or antigen(a bivalent monospecific of bispecific TFcA). The binding sites of aTFcBA may be of the same type or of a different type. For example, bothbinding sites may be TFcs, both binding sites may be Fabs, one bindingsite may be a Fab and the other binding site may be an scFv. Singledomain binding sites may also be used. A Fab will generally comprise aVH domain, which may be linked to a CH1 domain on the heavy chain of aTFcBA and a VL domain, which may be linked to a CL domain on the lightchain of the molecule. An scFv will generally comprise a VH domainlinked to an scFv linker that is linked to a VL domain

An scFv may be connected to a TFc by a connecting linker A connectinglinker may be about 1-5, 1-10, 1-15, 1-20 aas long or longer. Aconnecting linker is preferably chemically inert, non immunogenic andhas the required flexibility and rigidity for allowing the properconformation of a TFcBA comprising the scFv. In one embodiment, aconnecting linker comprises a Gly-Ser sequence, e.g., the aa sequence(G₄S)_(n), wherein n is 1, 2, 3, 4, or 5 or more. In one embodiment, aconnecting linker comprises (G₄S)₂ (see, e.g., FIG. 9).

An scFv comprises an scFv linker that links together the VH and the VLdomains. An scFv linker may be 15-30 or 20-25 aa long. An scFv linker ispreferably chemically inert, non immunogenic and has the requiredflexibility and rigidity for allowing the proper conformation of a TFcBAcomprising the scFv. In one embodiment, an scFv linker comprises aGly-Ser sequence, e.g., the aa sequence (G₄S)_(n), wherein n is 1, 2, 3,4, or 5 or more. However, other sequences may also be used. In certainembodiments, an scFv linker may comprise a portion of a hinge or a fulllength hinge alone or together with other aas, such as a (G₄S)_(n)sequence. In certain embodiments, an scFv linker comprises the sequence“AST” (the first 3 aa of a CH1 domain) upstream of a peptide linker,such as a Gly-Ser linker, e.g., (G₄S)₄ (see, e.g., FIG. 9).

In certain embodiments, a TFcA does not comprise a first and/or a secondhinge. Instead of a hinge, a TFcA may comprise a connecting linker Sucha linker may be a Gly-Ser linker as further described herein in thecontext of TFc linkers. An exemplary connecting linker may be shorterthan a TFc linker. In certain embodiments, a connecting linker comprisesa (G₄S)₃ or (G₄S)₄ sequence. In certain embodiments, a connecting linkercomprises a portion of a hinge, e.g., an upper hinge, middle hinge,lower hinge, or a combination thereof or a portion of one of these andanother peptide sequence, such as a (G₄S)_(n) sequence, wherein n is 1,2, 3, 4 or 5. Other peptide sequences may also be used as a connectinglinker provided that they provide the required flexibility and rigidityof certain parts of the linker

In certain embodiments, a binding site is an antigen binding site, suchas a Fab, scFv or single domain. Exemplary TFcAs comprise one or more VHand/or VL CDRs such as those from one or more of the variable regionsprovided herein. In certain embodiments, an anti-c-Met binding sitecomprises a VHCDR3 and/or a VLCDR3 sequence set forth in FIG. 9, such asthose of the variable domains of the humanized antibody 5D5(US2006/0134104) or the anti-c-Met binding site 2 (see Example 3). Incertain embodiments, an anti-c-Met binding site comprises 1, 2 or 3 CDRsof one of the VH domains set forth in FIGS. 9 and/or 1, 2 or 3 CDRs ofthe VL domain set forth in FIG. 9. In certain embodiments, an anti-EGFRbinding site comprises a VHCDR3 and/or a VLCDR3 sequence set forth inFIG. 9. In certain embodiments, an anti-EGFR binding site comprises 1, 2or 3 CDRs of one of the VH domains set forth in FIGS. 9 and/or 1, 2 or 3CDRs of one of the VL domains set forth in FIG. 9. Binding sites mayalso comprise one or more CDRs set forth in FIG. 9, wherein 1, 2 or 3aas have been changed, e.g., substituted, added or deleted, providedthat the binding sites are still able to bind specifically to theirtarget.

In certain embodiments, TFcAs comprise one or more variable domains setforth in FIG. 9. For example, an anti-c-Met binding site may comprise aVH and/or VL sequence set forth in FIG. 9, such as the variable domainsof the humanized antibody 5D5 (US2006/0134104) or the anti-c-Met bindingsite 2. Exemplary anti-EGFR binding sites comprise a VH and/or a VLsequence set forth in FIG. 9, such as those of panitumumab, 2224,cetuximab or humanized cetuximab H1L1, H1L2, H2L1 or H2L2 (see Example3).

In certain embodiments, anti-c-Met/anti-EGFR TFcAs comprise ananti-c-Met Fab and an anti-EGFR scFv. Table 14 shows combinations ofCDRs or variable domains of each of the following anti-c-Met andanti-EGFR aa sequences that may be used for forming TFcAs. The Tableprovides a SEQ ID NO if the sequence is provided herein or a “yes” if acombination is possible, but the resulting aa sequence is notspecifically provided. A person of skill in the art will be able tocreate such a molecule without undue experimentation based on the factthat all the elements of such proteins and nucleotide sequences encodingsuch are provided herein.

TABLE 14 Heavy chains of exemplary TFcAs Anti-c-Met Fab Humanized 5D5Binding site 2 Anti- panitumumab SEQ ID NO: 235, yes EGFR 343, 225, 227scFv and 229 2224 SEQ ID NO: 239 yes cetuximab H1L1 SEQ ID NO: 260 SEQID NO: 291 cetuximab H1L2 SEQ ID NO: 281 yes cetuximab H2L1 SEQ ID NO:283 yes cetuximab H2L2 SEQ ID NO: 285 yes

Light chains that may be used with the heavy chains in Table 14 are thelight chains of the particular anti-c-Met Fab used in the TFcA. Forexample, a TFcA comprising a VH domain from humanized 5D5, e.g., TFcAscomprising any one of SEQ ID NOs:225, 227, 229, 235, 239, 260, 281, 283,285 and 342, may be used with a light chain comprising the VL domain ofhumanized 5D5, i.e., SEQ ID NO:231. A TFcA comprising a VH domain fromthe anti-c-Met binding site 2, e.g., TFcAs comprising SEQ ID NO:291, maybe used with a light chain comprising the VH domain of the anti-c-Metbinding site 2, e.g., the VL domain of SEQ ID NO:289.

Antigen binding sites, e.g., the ones described herein, may beengineered for enhanced stability, reduced heterogeneity, enhancedexpression, enhanced solubility or other desirable characteristic.Methods for engineering of antibody fragments, such as scFv, VH, VL, andFab with enhanced stability and increased expression are described,e.g., in US 2006/0127893 US 2009/0048122 and references therein.

Variable domains may differ from those set forth herein in, or in atmost, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 50, 100 aas,provided that a binding site with a modified variable region retains itsability to bind specifically to its target antigen, e.g., a humanantigen selected from c-MET, ErbB2, ErbB3, ErbB4, IGF1R, IGF2R, Insulinreceptor, RON, EGFR, VEGFR1, VEGFR2, TNFR, FGFR1-4, PDGFR (alpha andbeta), c-Kit, EPCAM and EphA2. Variable domains for use in TFcAs, e.g.,TFcBAs, may also comprise a VH or VL aa sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the VH or VL aasequence set forth in FIG. 9, provided that a binding site with amodified variable domain retains its ability to bind specifically to itstarget antigen.

Anti-c-MET and/or anti-EGFR TFcBAs may also comprise a binding site thatbinds to the same epitope on human c-Met or human EGFR as the bindingsites provided herein, such as the ones having sequences set forth inFIG. 9. Binding sites encompassed herein may also competitively block orcompete with the binding of a binding site described herein, such as theones having sequences set forth in FIG. 9. A TFcA comprising a bindingsite that competes with a binding site described herein for binding to atarget antigen or epitope include the binding sites that are capable ofdisplacing a reference binding site (e.g., as described herein), e.g.,when added in an ELISA after the reference binding site, as well asbinding sites that prevent a reference binding site from binding whenthe binding site is added after the reference binding site to an ELISA.

TFcAs may also comprise variable domains from anti-c-Met, anti-c-Kit,anti-ErbB2, anti-ErbB3, anti-ErbB4, anti-IGF1R, anti-IGF2R, anti-Insulinreceptor, anti-RON, anti-VEGFR1, anti-VEGFR2, anti-TNFR, anti-FGFR1,anti-FGFR2, anti-FGFR3, anti-FGFR4, anti-PDGFR alpha, anti-PDGFR beta,anti-EPCAM, anti-EphA2 or anti-EGFR antibodies known in the art. Knownanti-c-Met antibodies are set forth in U.S. Pat. No. 5,686,292, U.S.Pat. No. 7,476,724, WO 2004/072117, WO 2004/108766, WO 2005/016382, WO2005/063816, WO 2006/015371, WO 2006/104911, WO 2007/126799, and WO2009/007427. Exemplary known anti-EGFR antibodies include ABX-EGF(Abgenix) (Yang, X. D., et al., Crit. Rev. Oncol./Hematol. 38 (2001)17-23) and humanized ICR62 (WO 2006/082515). Exemplary anti-c-Kitantibodies are set forth in U.S. Pat. No. 7,915,391 and EP 0586445B1.Exemplary anti-ErbB2 antibodies are set forth in U.S. Pat. No. 5,821,337and U.S. Pat. No. 7,560,111. Exemplary anti-ErbB3 antibodies are setforth in U.S. Pat. No. 7,705,130, U.S. Pat. No. 7,846,440 and WO2011/136911. Exemplary anti-ErbB4 antibodies are set forth in U.S. Pat.No. 7,332,579 and US 2010/0190964. Exemplary anti-IGF1R antibodies areset forth in U.S. Pat. No. 7,871,611 and U.S. Pat. No. 7,700,742.Exemplary anti-Insulin receptor antibodies are set forth in Bhaskar V.et al, Diabetes. 2012 May; 61(5):1263-71. Exemplary anti-RON antibodiesare set forth in WO 2012/006341, US 2009/0226442, and U.S. Pat. No.7,947,811. Exemplary anti-VEGFR1 antibodies are set forth in WO2005/037235. Exemplary anti-VEGFR2 antibodies are set forth in U.S. Pat.No. 8,057,791 and U.S. Pat. No. 6,344,339. Exemplary anti-TNFR1antibodies are set forth in EP 1972637B1 and US 2008/0008713. Exemplaryanti-FGFR1 antibodies are set forth in Ronca R et al, Mol Cancer Ther;9(12); 3244-53, 2010, and WO 2005/037235. Exemplary anti-FGFR2antibodies are set forth in WO 2011/143318. Exemplary anti-FGFR3antibodies are set forth in WO 2010/002862 and EP 1423428B1. Exemplaryanti-FGFR4 antibodies are set forth in WO 03/063893, WO 2008/052796 andUS 2010/0169992. Exemplary anti-PDGFR alpha antibodies are set forth inU.S. Pat. No. 8,128,929 and WO 1995/000659. Exemplary anti-PDGFR betaantibodies are set forth in U.S. Pat. No. 7,740,850. Exemplaryanti-EPCAM antibodies are set forth in U.S. Pat. No. 7,976,842, US2003/0157054, and WO 2001/007082. Exemplary anti-EphA2 antibodies areset forth in EP 1575509B1, U.S. Pat. No. 7,402,298, and U.S. Pat. No.7,776,328. Exemplary CD-44m antibodies are set forth in U.S. Pat. No.8,071,072, WO 2008/079246, U.S. Pat. No. 6,972,324. Exemplary CEAantibodies are set forth in U.S. Pat. No. 7,626,011. Exemplary ALKantibodies are set forth in U.S. Pat. No. 6,696,548, U.S. Pat. No.7,902,340, and WO 2008/131575. Exemplary AXL antibodies are set forth inUS 2010/0330095, US 2012/0121587, and WO 2011/159980.

In another embodiment, a binding site is a binding peptide. c-Metbinding peptides are known e.g. from Matzke, A., et al., Cancer Res 65(14) (2005) 6105-10. And Tam, Eric, M., et al., J. Mol. Biol. 385(2009)79-90.

Binding sites preferably bind specifically to their target with Kd of10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹ M, or 10⁻¹⁰ M or an even lower Kd value, asmeasured, e.g., by surface plasmon resonance (e.g., using a BIAcoresystem).

TFcAs may bind specifically to any target protein, e.g., soluble ormembrane human target proteins. Exemplary target proteins include humanreceptor proteins selected from the group consisting of ErbB2, ErbB3,ErbB4, IGF1R, IGF2R, Insulin receptor, RON, VEGFR1, VEGFR2, TNFR,FGFR1-4, PDGFR (alpha and beta), c-Kit, c-Met, EPCAM and EphA2.

Exemplary Heavy and Light Chains

In one embodiment, a TFcA comprises a heavy chain and a light chain. Inone embodiment, an anti-c-Met/anti-EGFR TFcBA comprises a heavy chaincomprising an aa sequence set forth in FIG. 9 and/or a light chaincomprising an aa sequence set forth in Example 3.

TFcBAs may also comprise a heavy chain and/or a light chain thatcomprise an aa sequence that differs from an aa sequence set forth inFIG. 9 in, or in at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,50, 100, 200, or 300 aas, provided that the TFcBA has the desiredbiological characteristic(s), as further described herein. A TFcBAs mayalso comprise a heavy chain and/or a light chain comprising an aasequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99%identical to the aa sequence of a heavy chain or light chain of FIG. 9,wherein the TFcBA has the desired biological characteristic(s).

TFcBAs may also comprise more than 2 binding sites, in which case, theheavy chain will comprise 1, 2, 3, 4 or more VH domains, which may belinked to the N- and/or C-terminus of any one of the followingmolecules: Fab-TFc-scFv; Fab-TFc-Fab; scFv-TFc-scFv; and scFv-TFc-Fab.The additional binding sites may be either Fabs or scFvs.

Biological Activities of TFcAs

In certain embodiments, a TFcA, e.g., a TFcBA, binding to one or moretarget proteins inhibits signal transduction mediated by the one or moretarget proteins. For example, an anti-c-Met+anti-EGFR TFcBA may inhibitsignal transduction mediated through either or both of c-Met and EGFR.Inhibition of signal transduction may be evidenced, e.g., by inhibitionof phosphorylation of EGFR and ERK. In certain embodiments, a TFcAinhibits phosphorylation of c-Met, EGFR and/or ERK by at least 30%, 40%,50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99% or more, relative tophosphorylation in the absence of the TFcA, when determined, e.g., atthe end of the experiment, e.g., as set forth in the Examples. PreferredTFcAs inhibit c-Met and/or EGFR signal transduction, e.g., measured byinhibition of phosphorylation of c-Met and EGFR, nearly completely,e.g., by at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or99.5%.

Although the biological characteristics described in this section aredescribed mostly in the context of anti-c-Met/anti-EGFR TFcBAs, thedescription also applies to other TFcBAs, as well as monovalent TFcAs.

Inhibition of a) ligand mediated phosphorylation of c-Met, and b)ligand-mediated phosphorylation of EGFR can be demonstrated by theability of a TFcBA to reproducibly decrease the level of phosphorylationof a) c-Met induced by an HGF family ligand, b) EGFR induced by an EGFRligand, e.g., EGF, or c) ERK induced by a c-Met ligand or an EGFRligand, each relative to the phosphorylation in control cells that arenot contacted with the TFcBA. The cell which expresses c-Met and/or EGFRcan be a naturally occurring cell or a cell of a cell line or can berecombinantly produced by introducing nucleic acid encoding c-Met and/orEGFR into a host cell. In certain embodiments, a TFcBA inhibits a HGFfamily ligand mediated phosphorylation of c-Met by at least about 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99%, or more, as determined, for example, byELISA, and calculated as set forth in the Examples. In certainembodiments, a TFcBA inhibits EGF-mediated phosphorylation of EGFR by atleast about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or more, as determined, forexample, by ELISA, and calculated as set forth in the Examples. Incertain embodiments, a TFcBA inhibits EGF and/or c-Met-mediatedphosphorylation of ERK by at least about 5%, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%,or more, as determined, for example, by ELISA, and calculated as setforth in the Examples.

TFcBAs may inhibit ligand induced phosphorylation of c-Met by at least70% or 80%, ligand induced phosphorylation of EGFR by at least 85%, 90%or 95% and optionally ligand induced phosphorylation of ERK by at least5% or 10%. TFcBAs may also inhibit ligand induced phosphorylation ofc-Met by at least 85%, ligand induced phosphorylation of EGFR by atleast 85% and optionally ligand induced phosphorylation of ERK by atleast 5%. In certain embodiments, TFcBAs inhibit ligand inducedphosphorylation of c-Met by at least 50% and ligand inducedphosphorylation of EGFR by at least 90%, and optionally ligand inducedphosphorylation of ERK by at least 5%.

TFcBAs may also be defined by the EC50 (i.e. the concentration of TFcBAat which 50% of maximum inhibition is obtained) of their inhibition ofphosphorylation of one or more of c-Met, EGFR and ERK, which EC50s maybe determined as further described herein. For example, TFcBAs disclosedherein may inhibit phosphorylation of c-Met with an EC50 of 10⁻⁸ M, 10⁻⁹M, 10⁻¹⁰ M or lower. They may inhibit phosphorylation of EGFR with anEC50 of 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M or lower. They may inhibitphosphorylation of ERK with an EC50 of 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ Mor lower. Some TFcBAs disclosed herein inhibit phosphorylation of c-Metby at least 80% or 85% with an EC50 of 10⁻⁸M, 10⁻⁹ M, 10⁻¹⁰ M or lower;inhibit phosphorylation of EGFR by at least 80% or 85% with an EC50 of10⁻⁸M, 10⁻⁹ M, 10⁻¹⁰ M or lower; and optionally inhibit phosphorylationof ERK by at least 5% with an EC50 of 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M orlower. In some cases, essentially complete blockage of either or both ofphosphorylation of c-Met and phosphorylation of EGFR will be obtainablewith a TFcBA herein disclosed.

In certain embodiments, a solution comprising TFcAs at a concentrationof 0.3, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 mg/ml ormore (or ranges of concentrations between any of these two numbers)comprises more than 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%of TFcBAs in unaggregated form (referred to in this context as monomers)as determined e.g., by Size Exclusion Chromatography (SEC) e.g.,following, a stability test as described below. The percentage ofmonomers may be determined in a solution after one of the followingstability tests: a) incubation at 4° C. for 1, 2, 3, 4, 5, or 6 days, or1, 2, 3 or more weeks; b) incubation at room temperature for 1, 2, 3, 4,5, or 6 days, or 1, 2, 3 or more weeks; c) incubation at 37° C. for 1,2, 3, 4, 5, or 6 days, or 1, 2, 3 or more weeks; d) 1, 2, 3, 4 or 5cycles of freeze/thaw, and e) agitation, e.g., gentle agitation on theorbital shaker at room temperature, e.g., for 1, 2, 3, 4, 5 or morehours.

In certain embodiments, a TFcA exhibits a stability after 1, 2, 3, 4 or5 days of incubation in serum at 37° C. of at least 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98% or 99%, relative to its stability at day 0,where the stability of a protein is determined by, e.g., SEC or bymeasuring its ability to bind to one or more of its target antigensafter incubation.

In certain embodiments, a TFcA has a melting temperature (Tm) asdetermined e.g., by Differential Scanning Fluorimetry (DSF) of at least50° C., 55° C., 58° C. or 60° C., as described in the Examples.

TFcAs may have a combination of two or more of the characteristics setforth herein. For example, a TFcBA may inhibit ligand inducedphosphorylation of c-Met by at least 70% and ligand inducedphosphorylation of EGFR by at least 70%, and also exhibit one or more ofthe following characteristics: (i) a Tm, as determined by DSF, of atleast 55 or 60° C.; and (ii) be at least 70%, 80% or 90% monomeric inPBS at 10 mg/mL after 5 or more days at at room temperature, 2 weeks at4° C., one or more cycles of freeze-thaw or gentle agitation. In certainembodiments, TFcBAs have a Tm of at least 60° C. and stability at roomtemperature, 4° C. or 37° C. of at least 90% (concentration of monomerafter incubation under these conditions relative to the initialconcentration of monomer).

In certain embodiments, a TFcA composition comprises one or more of thefollowing characteristics: 1) at least 50%, 60%, 70%, 80%, 90% or moreof the proteins are visible on SDS

PAGE after purification on protein A; 2) at least 50%, 60%, 70%, 80%,90% or more of observed species on SDS-PAGE gels are of the correctmolecular weight; 3) the thermal stability profile, as measured byDifferential Scanning Fluorimetry, does not show molten globularbehavior; 4) does not comprise more than 50%, 60%, 70%, 80%, 90%, 95%,98% or 99% or more monomer as visualized by SEC; and 5) inhibits morethan 95% of EGF receptor signaling activated by addition of exogenousEGF ligand, as measured by pEGFR inhibition.

Standard assays may be used for determining the biological activity andcharacteristics of TFcAs, such as TFcBAs. Exemplary assays are providedin the Examples.

Nucleic Acids, Expression Vectors and Host Cells

Provided herein are nucleic acids, e.g., DNA and RNA, encoding thepolypeptides described herein. Exemplary nucleotide sequences providedherein are those encoding the aa sequences set forth in the Figures. Incertain embodiments, a nucleotide sequence encoding a heavy or lightchain of a TFcA is linked to a sequence that enhances or promotes theexpression of the nucleotide sequence in a cell to produce a protein.Such nucleic acids may be encompassed within a vector, e.g., anexpression vector.

For the purposes of being secreted, a heavy and/or light chain of a TFcApreferably comprises a signal sequence, which is normally cut off aftersecretion to provide a mature polypeptide. The following signalsequences may be used:

MGFGLSWLFLVAILKGVQC (SEQ ID NO:241): for use, e.g., in expressing heavychains; andMGTPAQLLFLLLLWLPDTTG (SEQ ID NO:243) for use, e.g., in expressing lightchains.

An exemplary nucleotide sequence encoding SEQ ID NO:241 isatgggcttcggactgtcgtggctttttctggtggcgattcttaagggggtccagtgc (SEQ IDNO:240) and an exemplary nucleotide sequence encoding SEQ ID NO:243 isatgggcacccccgcacagctcttgttcttgctgcttctttggctccctgacacaactggt (SEQ IDNO:242).

Nucleic acids, e.g., DNA, that comprise a nucleotide sequence that is atleast about 70%, 75%, 80%, 90%, 95%, 97%, 98% or 99% identical to anucleotide sequence encoding a polypeptide described herein or anucleotide sequence set forth herein, and which encode a heavy and orlight chain of a TFcA, or portion thereof, as further described herein,are also encompassed herein. Such nucleotide sequences may encode aprotein set forth herein or may encode a protein that is at least 70%,75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical or similar to aprotein set forth herein or a portion thereof (e.g., a domain), such asan aa sequence set forth in any one of the Figures.

Also encompassed herein are cells, e.g., host cells, comprising anucleic acid or a vector provided herein.

The TFcAs described herein may be produced by recombinant means. Methodsfor recombinant production are widely known in the state of the art andcomprise protein expression in prokaryotic and eukaryotic cells withsubsequent isolation of the antibody and usually purification to apharmaceutically acceptable purity. For the expression of the TFcAs in ahost cell, nucleic acids encoding the respective polypeptides, e.g.,light and heavy chains, are inserted into expression vectors by standardmethods. Expression is performed in appropriate prokaryotic oreukaryotic host cells like CHO cells, NSO cells, SP2/0 cells, HEK293cells, COS cells, PER.C6 cells, yeast, or E. coli cells, and the TFcA isrecovered from the cells (supernatant or cells after lysis). Generalmethods for recombinant production of antibodies are well-known in thestate of the art and described, for example, in the review articles ofMakrides, S. C., Protein Expr. Purif 17 183-202 (1999); Geisse, S., etal, Protein Expr. Purif. 8 271-282 (1996); Kaufman, R. J., MoI.Biotechnol. 16 151-161 (2000); Werner, R. G., Drug Res. 48 870-880(1998).

The TFcAs may be suitably separated from the culture medium byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography. DNA and RNAencoding the TFcAs are readily isolated and sequenced using conventionalprocedures. The hybridoma cells can serve as a source of such DNA andRNA. Once isolated, the DNA may be inserted into expression vectors,which are then transfected into host cells such as HEK 293 cells, CHOcells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of recombinant TFcAs in the host cells.

Aa sequence variants (or mutants) of the TFcAs may be prepared byintroducing appropriate nucleotide changes into the TFcA DNA, or bynucleotide synthesis.

“Host cell” denotes any kind of cellular system which can be engineeredto generate the TFcAs described herein. In one embodiment, HEK293 cellsand CHO cells are used as host cells. Expression in NSO cells isdescribed by, e.g., Barnes, L. M., et al, Cytotechnology 32 109-123(2000); Barnes, L. M., et al., Biotech. Bioeng. 73 261-270 (2001).Transient expression is described by, e.g., Durocher, Y., et al., Nucl.Acids. Res. 30 E9 (2002). Cloning of variable domains is described byOrlandi, R., et al., Proc. Natl. Acad. Sci. USA 86 3833-3837 (1989);Carter, P., et al., Proc. Natl. Acad. Sci. USA 89 4285-4289 (1992); andNorderhaug, L., et al., J. Immunol. Methods 204 77-87 (1997). Anexemplary transient expression system (HEK 293) is described bySchlaeger, E.-J., and Christensen, K., in Cytotechnology 30 71-83 (1999)and by Schlaeger, E.-J., in J. Immunol. Methods 194 191-199 (1996).

The control sequences that are suitable for prokaryotes, for example,include a promoter, optionally an operator sequence, and a ribosomebinding site. Eukaryotic cells are known to utilize promoters, enhancersand polyadenylation signals.

A nucleic acid is “operably linked” when it is placed in a functionalrelationship with another nucleic acid sequence. For example, DNA for apre-sequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a pre-protein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading frame. However,enhancers do not have to be contiguous Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

Purification of TFcAs may be performed in order to eliminate cellularcomponents or other contaminants, e.g. other cellular nucleic acids orproteins, by standard techniques, including alkaline/SDS treatment, CsClbanding, column chromatography, agarose gel electrophoresis, and otherswell known in the art. See Ausubel, F., et al., ed. Current Protocols inMolecular Biology, Greene Publishing and Wiley Interscience, New York(1987). Different methods are well established and widespread used forprotein purification, such as affinity chromatography with microbialproteins (e.g. protein A or protein G affinity chromatography), ionexchange chromatography (e.g. cation exchange (carboxylmethyl resins),anion exchange (amino ethyl resins) and mixed-mode exchange), thiophilicadsorption (e.g. with beta-mercaptoethanol and other SH ligands),hydrophobic interaction or aromatic adsorption chromatography (e.g. withphenyl-sepharose, aza-arenophilic resins, or m-aminophenylboronic acid),metal chelate affinity chromatography (e.g. with Ni(II)- andCu(II)-affinity material), size exclusion chromatography, andelectrophoretical methods (such as gel electrophoresis, capillaryelectrophoresis) (Vijayalakshmi, M. A. Appl. Biochem. Biotech. 75 93-102(1998)).

Methods of Using TFcAs

Provided herein are methods of using TFcAs, e.g., TFcBAs. The TFcBAs canbe used for treating a disease or disorder associated with receptordependent signaling, including a variety of cancers.

In one embodiment, a method is provided for inhibiting proliferation ofa tumor cell expressing the targets of a TFcA, e.g., c-Met, ErbB2,ErbB3, ErbB4, IGF1R, IGF2R, Insulin receptor, RON, EGFR, VEGFR1, VEGFR2,TNFR, FGFR1-4, PDGFR (alpha and beta), c-Kit, EPCAM and/or EphA2. Amethod may comprise contacting the tumor cell with a TFcA such thatproliferation of the tumor cell is inhibited, slowed down, or stopped orsuch that the tumor cell dies.

Provided herein are methods for treating a disease or disorderassociated with the signaling pathway of the targets of a TFcBA, e.g.,c-Met, ErbB2, ErbB3, ErbB4, IGF1R, IGF2R, Insulin receptor, RON, EGFR,VEGFR1, VEGFR2, TNFR, FGFR1-4, PDGFR (alpha and beta), c-Kit, EPCAMand/or EphA2, by administering to a patient a TFcBA in an amounteffective to treat the disease or disorder. Suitable diseases ordisorders include, for example, a variety of cancers including, but notlimited to breast cancer and those set forth below. In one embodiment, amethod for treating a subject having a proliferative disease, such ascancer, comprises administering to a subject in need thereof atherapeutically effective amount of one or more TFcA.

Also provided is a method for (or a TFcA, e.g., a medicament for)treating a tumor expressing the target(s) of a TFcA, e.g., a TFcBA,e.g., c-Met, ErbB2, ErbB3, ErbB4, IGF1R, IGF2R, Insulin receptor, RON,VEGFR1, VEGFR2, TNFR, FGFR1-4, PDGFR (alpha and beta), c-Kit, EPCAM,EphA2 and/or EGFR, in a patient, the method comprising administering anamount of a TFcA effective to slow down or stop tumor growth, to stop orto shrink a tumor or to slow or stop tumor invasiveness or tumormetastasis). A tumor expressing c-Met, ErbB2, ErbB3, ErbB4, IGF1R,IGF2R, Insulin receptor, RON, VEGFR1, VEGFR2, TNFR, FGFR1-4, PDGFR(alpha and beta), c-Kit, EPCAM, EphA2 and/or EGFR may be treatedincluding tumors of the following cancers: gastric, esophageal,colorectal, non-small cell lung, pancreatic, prostate, renal, andthyroid cancers, hepatocellular carcinoma, glioma/glioblastoma, andbreast cancer (basal/triple-negative and HER2+).

A method of treating a tumor or a subject having a tumor can furthercomprise administering a second anti-cancer agent in combination withthe TFcA. Thus novel compositions are contemplated comprising a TFcA,together with a second anti-cancer agent, typically a biologic agenttogether with at least one pharmaceutically acceptable carrier orexcipient.

Also provided are kits comprising one or more TFcAs. The kits mayinclude a label indicating the intended use of the contents of the kitand optionally including instructions for use of the kit in treating adisease or disorder associated with a target of a TFcA dependentsignaling, e.g., EGFR and/or c-Met dependent signaling. The term labelincludes any writing, marketing materials or recorded material suppliedon or with the kit, or which otherwise accompanies the kit

Pharmaceutical Compositions

In another aspect, a composition, e.g., a pharmaceutical composition, isprovided for treatment of a tumor in a patient, as well as methods ofuse of each such composition to treat a tumor in a patient. Thecompositions provided herein contain one or more of the antibodies,e.g., TFcAs, disclosed herein, formulated together with apharmaceutically acceptable carrier. As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like that are physiologically compatible.Preferably, the carrier is suitable for intravenous, intramuscular,subcutaneous, parenteral, spinal or epidermal administration (e.g., byinjection or infusion). Depending on the route of administration, theantibody may be coated in a material to protect it from the action ofacids and other natural conditions that may inactivate proteins.

Pharmaceutical compositions may be administered alone or in combinationtherapy, i.e., combined with other agents. For example, the combinationtherapy can include an antibody of the present disclosure with at leastone additional therapeutic agent, such as an anti-cancer agent.Pharmaceutical compositions can also be administered in conjunction withanother anti-cancer treatment modality, such as radiation therapy and/orsurgery.

A composition of the present disclosure can be administered by a varietyof methods known in the art. As will be appreciated by the skilledartisan, the route and/or mode of administration will vary dependingupon the desired results.

To administer a composition provided herein by certain routes ofadministration, it may be necessary or desirable to coat the antibodywith, or co-administer the antibody with, a material to prevent itsinactivation. For example, the antibody may be administered to a patientin an appropriate carrier, for example, in liposomes, or a diluent.Pharmaceutically acceptable diluents include saline and aqueous buffersolutions. Liposomes include water-in-oil-in-water CGF emulsions as wellas conventional liposomes.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any excipient, diluent or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositionsprovided herein is contemplated. Supplementary active compounds (e.g.,additional anti-cancer agents) can also be incorporated into thecompositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. Saline solutions and aqueousdextrose and glycerol solutions can be employed as liquid carriers,particularly for injectable solutions. The composition, if desired, canalso contain minor amounts of wetting or solubility enhancing agents,stabilizers, preservatives, or pH buffering agents. In many cases, itwill be useful to include isotonic agents, for example, sodium chloride,sugars, polyalcohols such as mannitol, sorbitol, glycerol, propyleneglycol, and liquid polyethylene glycol in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

EXAMPLES

The following examples should not be construed as limiting the scope ofthis disclosure.

Throughout the examples, the following materials and methods are usedunless otherwise stated. In general, the practice of the techniques ofthe present disclosure employs, unless otherwise indicated, conventionaltechniques of chemistry, molecular biology, recombinant DNA technology,immunology (especially, e.g., antibody technology), pharmacology,pharmacy, and standard techniques in polypeptide preparation.

Example 1 Identification of Stable Tandem Fc Structures

This example describes the identification of stable multivalent Abformats. In this Example and in Example 2, protein constructs were usedthat do not contain binding sites. Several formats were compared, andeach of these formats was derived from either one of the following twotandem Fc constructs:

1) TFc “23” or “IgG1 TFc,” which comprises an IgG1 TFc comprising anIgG1 hinge; an IgG1 CH2 domain comprising the substitution N297Q; anIgG1 CH3 domain comprising the substitutions T366S/L368A/Y407V; a TFclinker consisting of (G4S)8, an IgG1 hinge that does not comprise theupper hinge; an IgG1 CH2 domain comprising the substitution N297Q; andan IgG1 CH3 domain comprising the substitution T366W. This constructcomprises the aa sequence set forth as SEQ ID NO:293 (see FIG. 11); and2) TFc “39” or “IgG1/IgG4 TFc,” which comprises an IgG1/IgG4 tandem TFccomprising a hybrid IgG1/IgG4 hinge comprising an IgG1 upper hinge and acore and lower IgG4 hinge; an IgG4 CH2 domain comprising thesubstitution T299K; an IgG1 CH3 domain comprising the substitutionsT366S/L368A/Y407V; a TFc linker consisting of (G4S)8; an IgG4 hinge thatdoes not comprise the upper hinge; an IgG4 CH2 domain comprising thesubstitution T299K; and an IgG1 CH3 domain comprising the substitutionT366W. This construct comprises the aa sequence set forth as SEQ IDNO:319 (see FIG. 11).

Six modified versions of TFc 23 and 39 were created, and these arelisted in Table 15. Briefly, a first modification was the addition of adisulfide bond in the viscinity of the knob or hole (TFc 23A). Anothermodification was the change of the knob hole of 23A for a smallerknob/hole (TFc 23B). Another modification introduced 1 or 2 cysteines inthe upper hinge of the first hinge, to create disulfide bridges withinthe TFc (TFc 23 D and C, respectively). Another modification introduceda C-terminal cysteine in the CH3 domain (TFc 23E). Another modificationin the TFc consisted of reducing the length of the TFc linker by 20 aas(TFc 23F, also referred to as “23G”). The aa sequences of these newlymodified TFcs (shown in Table 15) are the same as those set forth inFIGS. 6 and 7, except that the TFcs used in this example did notcomprise part of the upper hinge, i.e., aas EPKSC, and comprised asignal peptide. The nucleotide and aa sequences of these TFcs are shownin FIG. 11, and the identity of each of the domains or elements of theTFcs is set forth in Tables 12 and 13, with the only difference that thefirst hinge does not comprise EPKSC at its N-terminus

TABLE 15 TFcs Name of TFc SEQ ID NO TFc Modifications to TFc linker ofTFc 23 T366S/L368A/Y407V::T366W (G₄S) ₈ SEQ ID NO: 293 39 SEQ ID NO: 31923A Y349C/T366S/L368A/Y407V:: (G₄S) ₈ SEQ ID NO: 295 39A S354C/T366W SEQID NO: 321 23B Y407T::T366Y (G₄S) ₈ SEQ ID NO: 297 39B SEQ ID NO: 32323C H224C/T225C/T366S/L368A/ (G₄S) ₈ SEQ ID NO: 299 39C Y407V::T366W SEQID NO: 325 23D T223C/T366S/L368A/Y407V:: (G₄S) ₈ SEQ ID NO: 301 39DT366W SEQ ID NO: 327 23E T366S/L368A/Y407V/ (G₄S) ₈ SEQ ID NO: 303 39EC-term Cysteine::T366W/ SEQ ID NO: 329 C-term Cysteine 23G*T366S/L368A/Y407V::T366W (G₄S) ₄ SEQ ID NO: 305 *23G contains the sameTFc as that referred to elsewhere as “23F.”

The different nucleic acids (having SEQ ID NOs: 292, 294, 296, 298, 300,302, 304, 318, 320, 322, 324, 326, or 328) were transiently transfectedinto Freestyle 293F cells (Invitrogen) and purified with a one stepprotein A purification essentially as follows. The nucleic acidsencoding the proteins are cloned as single proteins into the expressionplasmid using standard recombinant DNA techniques. An expression vectoremployed is pCEP4 (Invitrogen). Expression plasmids are transfectedusing Polyethylene imine (2.5 μg/ml culture) and DNA (1 μg/ml cellculture). Transfected cells are incubated at 37° C., 5% CO₂ for six daysand then harvested. All proteins are purified using protein A affinityprotocol, in accordance with manufacturer's instructions. The protein Aaffinity step is used to selectively and efficiently bind the fusionproteins out of harvested cell culture fluids (HCCF). This removes >95%of product impurities in a single step with high yields and highthroughput. The portion of desired molecular form for fusion proteinsafter this step was in the range of 60 to 98 percent. MABSELECT from GEis used as the Protein A affinity resin. The purified material wasconcentrated and dialyzed into PBS.

A) Percentage Monomers

TFc solutions were subjected to the determination of the percentagemonomer present by Size Exclusion Chromatography (SEC) either in theinitial solution or after incubation at 4° C., 37° C., after freeze-thawor after gentle agitation on the orbital shaker at room temperature. SECwas performed essentially as follows. SEC is performed using Agilent1100 Series HPLC system. 50 μg of each molecule is injected on a TSKSuper SW3000 gel column (Tosoh Biosciences, P/N 18675). PBS is used asrunning and equilibration buffer at a flow rate of 0.35 ml/min

Table 16 provides the percentage monomer of exemplary TFcs in initialsolutions at the indicated concentrations.

TABLE 16 Percentage monomer of exemplary TFcs % monomer ProteinConcentration at 0 days 23G 3 mg/ml 87.9 23 5 mg/ml 71.4 39C 0.5 mg/ml39.5 39D 0.5 mg/ml 50.9 39 12.5 mg/ml 66.4

In another experiment, the percentage monomer was determined in theinitial solution after concentration of the molecules essentially asdescribed above. Table 17 provides the results.

TABLE 17 Percentage monomer in initial solution after concentrationmg/ml % monomer 23A 5.2 67% 23D 10.0 78% 23E 16.8 74%

A compilation of Tables 16 and 17 is shown below in Table 18

TABLE 18 Percentage monomer of exemplary TFcs Initial % monomer Second %monomer Concentration at initial concentration at second Protein (mg/ml)concentration (mg/ml) concentration 23 5.0 71.4% 23A 0.23 83.7% 5.2 67%23B 1.22 58.2% 23C 5.0 74.6% 23D 0.32 82.0% 10.0 78% 23E 0.74 77.5% 16.874% 23G 0.27 87.9% 39 12.5 66.4% 39A n/d n/d 39B 0.95 55.0% 39C 0.574.6% 39D 0.5 50.9% 39E 0.95 55.0% 39G 0.27 74.8%

Table 19 shows the percentage monomer of TFcs 39E and 23C in solution asdetermined after having been exposed to various conditions.

TABLE 19 Percentage monomer of 39E and 23C after exposure to variousconditions 39E 12.9 23C Condition mg/ml 5 mg/ml 0 days 89.7% 74.6% 4°C., 2 weeks 90.6% 82.5% Room temp., 90.3% 83.0% 10 days 37° C., 2 weeks88.1% — Freeze/thaw 90.9% 85.0% Agitate 90.7% —

The results indicate that the TFcs have very different stabilities inthe solution at day 0 and under the various conditions tested. 39E and23G appear to have better stability than others.

B) SDS PAGE Analysis

The TFcs were run on a 4-12% SDS-PAGE gel in non-denaturing conditionsand visualized by Coomassie stain. The results are shown in FIG. 8.

Example 2 Synthesis of Second Generation TFcs

To further improve the characteristics of the TFc molecules, furthermodifications were made to them. The modifications included (i) varyingthe length of the TFc linker (“23E (35L)”, “39E (35L)”, “23E (30L)”,“39E (30L)”, “23E (25L)” and “39E (25L)”); (ii) changing the combinationof AEMs and C-terminal cysteine modifications within each of the two CH3domains (“23E (35L) Inverted” and (“39E (35L) Inverted”); and (iii)changing the mutations that enhance CH3 association (“231”, “391”, “23J”and “39J”). These modifications are summarized in Table 20. The aasequences of these newly modified TFcs are set forth in FIG. 11 and theidentity of each of their domains or elements is set forth in Tables 12and 13.

TABLE 20 Second generation TFcs Name of TFcBA Modifications to TFc TFclinker SEQ ID NO of TFc 23E (35L) T366S/L368A/Y407V/C-term Cysteine(G4S) ₇ SEQ ID NO: 185 39E (35L) KSCDKT::T366W/C-term Cysteine GEC SEQID NO: 211 23E (35L) T366S/L368A/Y407V/C-term Cysteine (G4S) ₇ SEQ IDNO: 187 Inverted 39E GEC::T366W/C-term Cysteine KSCDKT SEQ ID NO: 213(35L) Inverted 23E (30L) T366S/L368A/Y407V/C-term Cysteine (G4S) ₆ SEQID NO: 189 39E (30L) KSCDKT::T366W/C-term Cysteine GEC SEQ ID NO: 21523E (25L) T366S/L368A/Y407V/C-term Cysteine (G4S) ₅ SEQ ID NO: 191 39E(25L) KSCDKT::T366W/C-term Cysteine GEC SEQ ID NO: 217 231S364H/F405A::Y349T/T394F (G4S) ₈ SEQ ID NO: 193 391 SEQ ID NO: 219 23JK370D/K392D/K409D::E356K/E357K/ (G4S) ₈ SEQ ID NO: 195 23J D399K SEQ IDNO: 221

The second generation TFcs were expressed and purified essentially asdescribed in Example 1.

A) Percentage Monomers

TFc solutions were subjected to the determination of the percentagemonomer present by SEC either in the initial solution or after 7 days at4° C. SEC was performed essentially as described in Example 1.

Table 21 provides the percentage monomer of exemplary second generationTFcs in initial solutions and after 7 days at 4° C.

TABLE 21 Percentage monomer of exemplary TFcs Day 1 Day 7 Fc1 23E/25L74.8% Fc2 23E/35L Not measured 77.9% Fc3 39E/30L 80.2% Fc4 39E/35L Notmeasured 78.9% Fc5 39E/35L inv 79.3% Fc6 23E/30L 80.2% Fc7 23E/35L inv79.1% Fc8 39E/25L 78.1% Original IgG1 Fc 23E/40L 78.8% Original IgG1/4Fc 39E/40L 88.3%

The results indicate that a 40 aa linker, e.g., in molecules 23E and39E, results in a more stable TFc than a shorter linker

Example 3 Exemplary Anti-c-Met/Anti-EGFR TFcBAs

A) Exemplary Anti-c-Met Binding Sites

A TFcBA may comprise an anti-c-Met binding site comprising or consistingof that of the humanized 5D5 Ab (U.S. Pat. No. 7,476,724). The heavychain may comprise the following Fab domain or VH domain thereof:

1) Without signal peptide:

STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV(SEQ ID NO: 223; the CDRs are underlined with a dotted line, andthe CH1 domain is underlined)2) Including the exemplary signal peptide consisting of SEQ ID NO: 241 (underlined):

NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV(SEQ ID NO: 245; the signal peptide is underlined and boldface,the CDRs are underlined with a dotted line, and the CH1 domainis underlined)

The light chain may comprise the following Fab domain or VH domainthereof:

1) Without signal peptide:

PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 231; the CDRs are underlined with a dotted line, andthe CL domain is underlined)2) Including the exemplary signal peptide consisting of SEQ ID NO: 243:

GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 247; the signal peptide is underlined and boldface,the CDRs are underlined with a dotted line, and the CL domain isunderlined)

A TFcBA may also comprise an anti-c-Met binding site comprising orconsisting of the following heavy and light chain portions, and referredto herein as “anti-c-Met binding site 2.” The heavy chain may comprisethe following Fab domain or VH domain thereof:

1) Without signal peptide:

STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV(SEQ ID NO: 287; the CDRs are underlined with a dotted line,and the CH1 domain is underlined)2) Including the exemplary signal peptide consisting of SEQ ID NO:241 (underlined):

SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV(SEQ ID NO: 256; the signal peptide is underlined and boldface;the CDRs are underlined with a dotted line, and the CH1 domainis underlined)

The light chain may comprise the following Fab domain or VH domainthereof:

1) Without signal peptide:

DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 289; the CDRs are underlined with a dotted line, andthe CL domain is underlined)2) Including the exemplary signal peptide consisting of SEQ ID NO: 243:

GGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 345; the signal peptide is underlined and boldface;the CDRs are underlined with a dotted line, and the CL domainis underlined)

The aa sequences of the heavy chain of an exemplary mature TFcBAcomprising in amino to carboxy terminal order: i) the Fab domain of theanti-c-Met binding site 5D5 (SEQ ID NO:223); ii) a TFc comprising AEM 1and DiS 2 (SEQ ID NO:181); and iii) the panitumumab anti-EGFR scFv H1L1having SEQ ID NO:233 (see below) is set forth as SEQ ID NO:235 (FIG. 9).The aa sequences of the heavy chain of an exemplary mature TFcBAcomprising in amino to carboxy terminal order: i) the Fab domain of theanti-c-Met binding site 2 (SEQ ID NO:287), ii) a TFc comprising AEM 1and DiS 2 (SEQ ID NO:181); and iii) the cetuximab anti-EGFR scFv H1L1having SEQ ID NO:258 (see below) is set forth as SEQ ID NO:291 (FIG. 9).Generally, the exemplary anti-c-Met Fab heavy chain sequences providedhere may be linked to any of the TFcs, or constructs comprising a TFc,disclosed herein. These proteins may be expressed with a signalsequence, which may be the signal sequence consisting of SEQ ID NO:241.

B) Exemplary Anti-EGFR scFvs

A TFcBA may comprise any of the following anti-EGFR scFvs (or variabledomains or CDRs thereof):

1) Panitumumab (VECTIBIX) scFvThe aa sequence of the VH and VL domains of panitumumab is providedin U.S. Pat. No. 6,235,883, and are assembled into an scFv having thefollowing aa sequence:

RT (SEQ ID NO: 233; the scFv linker is in italics and the VH and VL CDRs are underlined with a dotted line) 2) 2224 scFvThe aa sequence of the VH and VL domains of Ab 2224 is provided in US Patent Publication No 2010/0009390, and are assembled intoan scFv having the following aa sequences:

(SEQ ID NO: 237; the scFv linker is in italics and the VH and VL CDRs are underlined with a dotted line)3) Humanized Cetuximab scFv

The variable regions of Cetuximab were humanized and used forconstructing the following scFvs, wherein the CDRs are underlined withdotted lines, the scFv linker is italicized and aa modificationsresulting from the humanization are in lower case letters:

(SEQ ID NO: 258) 3.1) cetuximab scFv H1 L1

(SEQ ID NO: 275) 3.2) cetuximab scFv H1 L2

(SEQ ID NO: 277) 3.3) cetuximab scFv H2 L1

(SEQ ID NO: 279) 3.4) cetuximab scFv H2 L2

The VH and VL domains of the humanized cetuximab Abs may be used in anyother format of Ab, e.g., an Ab having a naturally occurring structurecomprising two heavy chains and two light chains.

The aa sequence of the heavy chain of an anti-c-Met/anti-EGFR TFcBAcomprising i) the humanized 5D5 anti-c-Met VH domain (SEQ ID NO:223);ii) a TFc comprising AEM 1 and DiS 2 (SEQ ID NO:181); and iii) thepanitumumab anti-EGFR scFv having SEQ ID NO:233 is set forth as SEQ IDNO:235 (FIG. 9). The aa sequences of the heavy chain ofanti-c-Met/anti-EGFR TFcBAs comprising the same binding sites as thosein SEQ ID NO:235, but comprising a different TFc are set forth in SEQ IDNOs: 343, 225, 227 and 229 (FIG. 9). The aa sequence of the heavy chainof an anti-c-Met/anti-EGFR TFcBA comprising i) the humanized 5D5anti-c-Met VH domain (SEQ ID NO:223); ii) a TFc comprising AEM 1 and DiS2 (SEQ ID NO:181); and iii) the 2224 anti-EGFR scFv having SEQ ID NO:237is set forth as SEQ ID NO:239 (FIG. 9). The aa sequences of the heavychain of an anti-c-Met/anti-EGFR TFcBA comprising i) the humanized 5D5anti-c-Met VH domain (SEQ ID NO:223); ii) a TFc comprising AEM 1 and DiS2 (SEQ ID NO:181); and iii) a cetuximab anti-EGFR scFv consisting of SEQID NO:258, 275, 277 or 279 are set forth as SEQ ID NOs:260, 281, 283 and285, respectively (FIG. 9). Generally any of the anti-EGFR scFvsdisclosed herein, or their variable or CDR sequences, may be linked toany of the TFcs, or constructs comprising a TFc, disclosed herein.

Nucleotide sequences encoding the Fab domains, scFvs and TFcBAs areprovided in FIG. 10.

Other exemplary anti-c-Met/anti-EGFR TFcBAs are set forth in Table 21,wherein each of the sequences may be connected to the adjacent sequencein amino to carboxy terminal order without intervening sequence.

TABLE 21 Exemplary TFcBAs Anti-c-Met Con- Anti- heavy chain necting EGFRFab TFc linker scFv Humanized 5D5 (SEQ IgG1 TFc (SEQ ID NO: 171, 173,175, (G4S)2 Panitumumab ID NO: 223 or 245) 177, 179, 181, 183, 185, 187,189, 191, (SEQ ID with light chain having 193 or 195) or IgG1/IgG4hybrid TFc NO: 233) SEQ ID NO: 231 or (SEQ ID NO: 197, 199, 201, 203,205, 247 207, 209, 211, 213, 215, 217, 219 or 221) Humanized 5D5 (SEQIgG1 TFc (SEQ ID NO: 171, 173, 175, (G4S)2 2224 ID NO: 223 or 245) 177,179, 181, 183, 185, 187, 189, 191, (SEQ ID with light chain having 193or 195) or IgG1/IgG4 hybrid TFc NO: 237) SEQ ID NO: 231 or (SEQ ID NO:197, 199, 201, 203, 205, 247 207, 209, 211, 213, 215, 217, 219 or 221)Humanized 5D5 (SEQ IgG1 TFc (SEQ ID NO: 171, 173, 175, (G4S)2 CetuximabID NO: 223 or 245) 177, 179, 181, 183, 185, 187, 189, 191, H1L1 withlight chain having 193 or 195) or IgG1/IgG4 hybrid TFc (SEQ ID SEQ IDNO: 231 or (SEQ ID NO: 197, 199, 201, 203, 205, NO: 258) 247 207, 209,211, 213, 215, 217, 219 or 221) Humanized 5D5 (SEQ IgG1 TFc (SEQ ID NO:171, 173, 175, (G4S)2 Cetuximab ID NO: 223 or 245) 177, 179, 181, 183,185, 187, 189, 191, H1L2 with light chain having 193 or 195) orIgG1/IgG4 hybrid TFc (SEQ ID SEQ ID NO: 231 or (SEQ ID NO: 197, 199,201, 203, 205, NO: 275) 247 207, 209, 211, 213, 215, 217, 219 or 221)Humanized 5D5 (SEQ IgG1 TFc (SEQ ID NO: 171, 173, 175, (G4S)2 CetuximabID NO: 223 or 245) 177, 179, 181, 183, 185, 187, 189, 191, H2L1 withlight chain having 193 or 195) or IgG1/IgG4 hybrid TFc (SEQ ID SEQ IDNO: 231 or (SEQ ID NO: 197, 199, 201, 203, 205, NO: 277) 247 207, 209,211, 213, 215, 217, 219 or 221) Humanized 5D5 (SEQ IgG1 TFc (SEQ ID NO:171, 173, 175, (G4S)2 Cetuximab ID NO: 223 or 245) 177, 179, 181, 183,185, 187, 189, 191, H2L2 with light chain having 193 or 195) orIgG1/IgG4 hybrid TFc (SEQ ID SEQ ID NO: 231 or (SEQ ID NO: 197, 199,201, 203, 205, NO: 279) 247 207, 209, 211, 213, 215, 217, 219 or 221)Binding site 2 (SEQ IgG1 TFc (SEQ ID NO: 171, 173, 175, (G4S)2Panitumumab ID NO: 287 or 256) 177, 179, 181, 183, 185, 187, 189, 191,(SEQ ID with light chain having 193 or 195) or IgG1/IgG4 hybrid TFc NO:233) SEQ ID NO: 289 or (SEQ ID NO: 197, 199, 201, 203, 205, 345 207,209, 211, 213, 215, 217, 219 or 221) Humanized 5D5 (SEQ IgG1 TFc (SEQ IDNO: 171, 173, 175, (G4S)2 2224 ID NO: 223 or 245) 177, 179, 181, 183,185, 187, 189, 191, (SEQ ID with light chain having 193 or 195) orIgG1/IgG4 hybrid TFc NO: 237) SEQ ID NO: 231 or (SEQ ID NO: 197, 199,201, 203, 205, 247 207, 209, 211, 213, 215, 217, 219 or 221) Humanized5D5 (SEQ IgG1 TFc (SEQ ID NO: 171, 173, 175, (G4S)2 Cetuximab ID NO: 223or 245) 177, 179, 181, 183, 185, 187, 189, 191, H1L1 with light chainhaving 193 or 195) or IgG1/IgG4 hybrid TFc (SEQ ID SEQ ID NO: 231 or(SEQ ID NO: 197, 199, 201, 203, 205, NO: 258) 247 207, 209, 211, 213,215, 217, 219 or 221) Humanized 5D5 (SEQ IgG1 TFc (SEQ ID NO: 171, 173,175, (G4S)2 Cetuximab ID NO: 223 or 245) 177, 179, 181, 183, 185, 187,189, 191, H1L2 with light chain having 193 or 195) or IgG1/IgG4 hybridTFc (SEQ ID SEQ ID NO: 231 or (SEQ ID NO: 197, 199, 201, 203, 205, NO:275) 247 207, 209, 211, 213, 215, 217, 219 or 221) Humanized 5D5 (SEQIgG1 TFc (SEQ ID NO: 171, 173, 175, (G4S)2 Cetuximab ID NO: 223 or 245)177, 179, 181, 183, 185, 187, 189, 191, H2L1 with light chain having 193or 195) or IgG1/IgG4 hybrid TFc (SEQ ID SEQ ID NO: 231 or (SEQ ID NO:197, 199, 201, 203, 205, NO: 277) 247 207, 209, 211, 213, 215, 217, 219or 221) Humanized 5D5 (SEQ IgG1 TFc (SEQ ID NO: 171, 173, 175, (G4S)2Cetuximab ID NO: 223 or 245) 177, 179, 181, 183, 185, 187, 189, 191,H2L2 with light chain having 193 or 195) or IgG1/IgG4 hybrid TFc (SEQ IDSEQ ID NO: 231 or (SEQ ID NO: 197, 199, 201, 203, 205, NO: 279) 247 207,209, 211, 213, 215, 217, 219 or 221)

Example 4 Methods for Preparing and Characterizing TFcAs or TFcs A)Protein Expression and Purification

Stable Transfection:

Nucleic acids are transfected into CHO-K1 cells (Chinese hamster ovary;ATCC cat #CCL-61) using 1:1(:1) plasmid ratio, and are purified with aone step protein A purification method, e.g., according to the followingprotocol. The nucleic acids encoding the TFcAs or TFcs are cloned assingle proteins into the expression plasmids using standard recombinantDNA techniques. An exemplary expression vector employed is pMP 10K(SELEXIS). Expression plasmids are linearized, purified using QIAquickpurification kit (QIAGEN), and co-transfected into CHO-K1 cells usingLipofectamine LTX (Invitrogen). Transfected cells are recovered withHam's F12 medium (Gibco) containing 10% FBS for 2 days without selectionpressure, then with selection pressure for 4 days. After 4 days, theyare changed into serum-free medium (Hyclone) containing glutamine withselection pressure. After a week, cells are checked for expression andscaled up to desired volume. All proteins are purified using protein Aaffinity protocol, carried out in accordance with manufacturer'sinstructions. The protein A affinity step is used to selectively andefficiently bind the TFcA or TFc proteins out of harvested cell culturefluids (HCCF). This removes >95% of product impurities in a single stepwith high yields and high throughput. The portion of desired molecularform for TFcAs or TFcs after this step is expected to be in the range of60 to 98 percent. MABSELECT from GE is used as the Protein A affinityresin. The purified material is concentrated and dialyzed into PBS.

Transient transfection: Nucleic acids are transiently transfected intoFreestyle 293F cells (Invitrogen) and purified with a one-step protein Apurification essentially as follows. The nucleic acids encoding theproteins are cloned as single proteins into the expression plasmid usingstandard recombinant DNA techniques. An exemplary expression vectoremployed is pCEP4 (Life Technologies cat #R790-07). Expression plasmidsare transfected using Polyethylene imine (2.5 μg/ml culture) and DNA (1μg/ml cell culture). Transfected cells are incubated at 37° C., 5% CO₂for six days and then harvested. All proteins are purified using proteinA affinity protocol, in accordance with manufacturer's instructions. Theprotein A affinity step is used to selectively and efficiently bind thefusion proteins out of harvested cell culture fluids (HCCF). Thisremoves >95% of product impurities in a single step with high yields andhigh throughput. MABSELECT from GE is used as the Protein A affinityresin. The purified material is concentrated and dialyzed into PBS.

B) SDS-PAGE Analysis

TFcBAs or TFcs are run on a 4-12% SDS-PAGE gel in non-denaturingconditions and visualized by Coomassie stain. This method may be used todetermine whether a TFcA or TFc is properly formed or assembled.

C) Thermal Stability Measurement by DSF

The temperature at which a TFcA or TFc unfolds is determined byDifferential Scanning Fluorimetry (DSF) essentially as follows. The DSFassay is performed in the IQ5 Real Time Detection System (Bio-Rad). 20μl solutions of 15 uM TFcA or TFc, 1× Sypro Orange (Invitrogen LifeTechnologies), and 1×PBS are added to the wells of a 96 well plate. Theplate is heated from 20° C. to 90° C. with a heating rate of 1° C./minData is transferred to GraphPad Prism for analysis.

D) pEGFR Inhibition

Inhibition of signal transduction through EGFR, e.g., ligand-inducedsignal transduction, by a TFcBA may be determined by measuring theeffect of the particular TFcA on the phosphorylation of EGFR.

The following protocol is used to measure inhibition of EGFR. Cells(e.g. A431 cells (ATCC cat #CRL-1555) or NCI-H322M (National CancerInstitute)) are maintained in DMEM medium supplemented with 10% fetalbovine serum, Penicillin/Streptomycin and L-glutamine. For signalingexperiments, 3.5×10⁴ cells are plated in complete medium in 96-welltissue culture plates. The following day, complete medium is replacedwith serum-free medium, and cells are incubated overnight at 37° C.Cells are pretreated for 2 hours with starting concentration of 300 nMand titrating 3-fold down for an 11 concentration dose for each TFcA orTFc, and then stimulated for 10 minutes with 8 nM EGF (Human recombinantEGF; Cat#AF-100-15; PeproTech, Inc.). Cells are washed with PBS andlysed in MPER buffer (cat #PI78505, VWR International) supplemented withprotease and phosphatase inhibitors (cOmplete Protease InhibitorCocktail Tablet provided in EASY packs, cat #4693124001, RocheDiagnostics Corp; PhosSTOP Phosphatase Inhibitor Cocktail Tablets, cat#4906837001, Roche Diagnostics Corp). ELISAs for phospho-EGFR (pEGFR)are performed according to the manufacturer's protocols (pEGFR ELISA R&Dkit (cat #: DYC1095-C)), with the exception that the capture Ab is EGFRAb-11, Clone: 199.12 (Fisher Scientific Cat# MS396P1ABX). SuperSignalELISA Pico Chemiluminescent Substrate (cat #PI37069, VWR International)is added and plates read on a PerkinElmer Envision plate reader.Luminescence values are plotted following normalization to the observedsignal at the lowest concentration of TFcA or TFc. For data analyses,duplicate samples are averaged and error bars are used to represent thestandard deviation between the two replicates. Inhibition curves andcorresponding IC50 values are calculated using GraphPad Prism software(GraphPad Software, Inc.) via regression of the data to a 4 parameterlogistic equation. To calculate percent inhibition, regressed values ofmaximal (‘max’) and minimum (‘min’) inhibitor potency can be utilized asfollows:

curve_span=max−min;baseline_span=max;percent_inhibition=100*curve_span/baseline_span.E) pERK Inhibition

Inhibition of signal transduction through c-Met and/or EGFR, e.g.,ligand-induced signal transduction, by a TFcA may may be determined bymeasuring the effect of the particular TFcA on the phosphorylation ofERK. The following protocol may be used to measure inhibition of pERK.Day 1: Actively midlog (about 80% confluency) growing cells (e.g., A431cells) are split in DMEM (+10% FBS+L/glutamine+Pen/Strep) media.Approximately 35,000 cells are seeded/well in a 96 well-plate. Day 2:The media is changed from 10% FBS to serum-free media−0.5% FBS(+L/glutamine+Pen/Strep) media. Day 3: The inhibitors/antibodies arediluted into the appropriate volume of serum media. 100 μL of eachinhibitor per concentration is added/well. The inhibitor is allowed toincubate at 37° C. for 2 hours. At the end of the 2 hour period, a finalconcentration of 8 nM EGF (Human recombinant EGF; Cat#AF-100-15;PeproTech, Inc.) is added to each inhibitor and each concentration ofinhibitor for 10 min. The cells are washed 2× with cold PBS and laterlysed in 40 μL/well of SureFire Lysis buffer (a 1:5 dilution of stockwith water). The lysates are place in −80° C. usually within 5 min afterlysis. Day 4: The protocol on performing the SureFire pERK 1/2 ELISA canbe found in Perkin Elmer website (ALPHASCREEN PROTEIN A 10K PTSPerkinElmer Life Sciences, Inc. Cat #: 6760617M; TGR Surefire ERK1 384Kit for 10,000 Ass; PerkinElmer Life Sciences, Inc. Cat #: TGRES10K).Essentially, the −80° C. lysate is thawed at room temperature. In themeantime, the reaction buffer is prepared in which the Activation Bufferand Reaction Buffer are mixed according to protocol. The Protein Adetection kit reagents are added last to the reaction buffer prior toadding onto the 384 well plate. 4 μL of the thawed lysate is transferredonto a ProxiPlate 384, white shallow well plate (from Perkin Elmer; cat#6008280). After addition of the Protein A detection kit reagents, 7 μLof the final reaction buffer is transferred to each well (which alreadyhas 4 μL of the lysate in them). The plates are sealed tightly withaluminum sealer. The plate is spun down in an Eppendorf table topcentrifuge at 1800 rpms for 1 minute. The plates are gently shaken at RTfor 2 hours. The plates are then read in Perkin Elmer Envision Reader.Normalization of luminescence data and calculation of IC50 occurs asdescribed for pEGFR.

Example 5 Protocols for Measurement of ELISA Plate-Based AntibodyBinding

Binding of Bispecific Antibodies to Soluble cMet-Fc and EGFR-his

Reacti-bind plates (96 well) are coated with 50 μL of cMet-Fc (2 μg/mLin PBS), and incubated overnight at 4° C. Next day, the plates arewashed with PBS-T (PBS+0.05% Tween-20), blocked for 1 hour at roomtemperature with 100 μL of blocking buffer, and washed again with PBS-T.Plates are incubated with 50 μL of bispecific antibodies at roomtemperature for 2 hours, and then washed with PBS-T. Antibodyconcentrations start at 500 nM (in PBS-T), and include ten additionaltwo-fold dilutions and one blank (PBS-T only). Plates are then incubatedwith 50 μL of EGFR-his (at 1 μg/ml in PBS-T for one hour at roomtemperature. The plates are washed with PBS-T and then incubated withanti-his-HRP antibody diluted 1:10,000 in PBS-T for 1 hour at roomtemperature, and washed again with PBS-T. The plates are incubated with100 μL of TMB substrate for 5-10 minutes at room temperature and thereaction is stopped by adding 100 μL of Stop solution. The absorbancewas measured at 450 nm, and the resulting data analyzed using GraphPadPrism.

Exemplary results using TFcBAs in the method above can be seen in FIG.15, which shows binding affinity of onartuzumab-39Egy-2224 (squares) andonartuzumab-39Egy4-panitumumab (circles).

Example 6 Current Technologies for Asymmetric Fc-Domains Give MolecularWeight Heterogeneity Stable Transfection of CHO-K1 Cells

Suspension-adapted CHO-K1 cells are grown in Hyclone Media supplementedwith 8 mM L-glutamine to a density of 2 million/mL. On the day oftransfection, the cells are resuspended in a serum-free media (Opti-MEMI) at a density of 80,000 cells/mL. The cells (500 μL) are thentransfected with 1 μg of total DNA (including 10 ng of pNeo vector, anin-house vector carrying the geneticin selection marker) using 2.75 μLof Lipofectamine in a 24 well plate. After 3 hours, 1 mL of recoverymedia (HAMS-F12+10% FBS) is added, and the transfected cells allowed torecover for 48 hours. The cells are then expanded into a 96-well plate,and the selection marker geneticin was added to the recovery media at500 μg/ml. After 4 more days, the media is replaced with serum freeHyclone media (supplemented with L-glutamine), and the transfected cellsallowed to adapt. After a week, the selected cells form colonies, andthe wells are tested for desired characteristics with western blots fromthe supernatant. The desired clones are expanded to a 24-well plate,then to a T-25 flask, and eventually to a shake flask. The desiredclones are confirmed with SDS-PAGE, and scaled up to the desired volume.The cells are harvested by centrifugation (6000 g, 30 min) when theviability falls below 80%, and the supernatant filtered using a 0.22 μmfilter.

Cells were transfected as described aboved and analyzed as follows.Results are shown in Table 22 below.

A) Western Blot Protocol

-   -   Cell supernatants expressing onartuzumab were run on a 4-12%        SDS-PAGE gel in non-denaturing conditions. The proteins were        transferred to nitrocellulose paper using the Invitrogen iBlot.        The blot was washed with PBS-T and then incubated for one hour        with anti-human-FC conjugated to IRD700. The blot was washed        three times with PBS-T and then imaged using the Li-Cor Odyssey.        The results are shown in FIG. 12.

B) Percentage Monomers

-   -   TFc solutions were subjected to the determination of the        percentage monomer present by SEC    -   in the initial solution. SEC was performed essentially as        described in Example 1.    -   Table 22 provides the percentage monomer of exemplary second        generation TFcs in initial solutions.

TABLE 22 Percentage monomer of exemplary onartuzumab clones Protein HighMW species % monomer Low MW species Onartuzumab1 78 22 Onartuzumab2 7525 Onartuzumab3 n/d Onartuzumab4 24 75 Onartuzumab5 89 11 Onartuzumab62.2 98

Example 7 Production and Analysis of Charged Aglycosylation Mutants

The identification of stable multivalent Ab formats is described below.Protein constructs were used that do not contain binding sites. Severalformats were compared as shown in Table 23 and FIG. 17.

TABLE 23 TFcs Name of Modifications to TFc SEQ ID NOs of tandem Fc CH2domain TFc (aa/nucleotide) Glyco_wt None 357/358 Glyco_1N297D/T299S::N297D/T299S 388/389 Glyco_2 T299K::N297D/T299S 390/391Glyco_3 N297D/T299S::T299K 392/393 Glyco_4 N299K::N299D 394/395 Glyco_5N299D::N299K 396/397 Glyco_6 N299D::N299D 398/399

Nucleic acids (having SEQ ID NOs:357, and 389-399 (odd numbers) weretransiently transfected into Freestyle 293F cells and purified with aone-step protein A purification followed by DSF essentially as describedin Example 4.

TABLE 24 Percentage monomer of TFcs after exposure to various conditionsProtein Condition mg/ml % monomer Glyco_wt Initial purification 10.686.6 4 deg, 0 day 10.6 79.7 Agitate 10.6 79.3 4 deg, 10 day 10.6 78.6 37deg, 10 day 10.6 75.5 4 deg, 1 month 10.6 77.5 Glyco_1 Initialpurification 3.7 83.2 4 deg, 0 day 11.2 83.0 Agitate 11.2 82.6 4 deg, 10day 11.2 82.4 37 deg, 10 day 11.2 80.3 4 deg, 1 month 11.2 84.7 Glyco_2Initial purification 1.3 78.6 4 deg, 0 day 8.4 78.0 Agitate 8.4 n/dGlyco_3 Initial purification 3 76.4 4 deg, 0 day 12 75.0 Agitate 12 n/d4 deg, 10 day 12 73.5 37 deg, 10 day 12 71.6 Glyco_4 Initialpurification 4.2 84.5 4 deg, 0 day 10.1 83.5 Agitate 10.1 83.6 4 deg, 10day 10.1 83.0 37 deg, 10 day 10.1 80.8 Glyco_5 Initial purification 9.577.1 4 deg, 0 day 9.5 76.3 Agitate 9.5 76.5 4 deg, 10 day 9.5 76.6 37deg, 10 day 9.5 73.7 4 deg, 1 month 9.5 76.5 Glyco_6 Initialpurification 6.3 81.7 4 deg, 0 day 12 80.6 Agitate 12 81.2 4 deg, 10 day12 81.0 37 deg, 10 day 12 77.3 4 deg, 1 month 12 80.8

B) SDS PAGE Analysis

The TFcs were run on a 4-12% SDS-PAGE gel in denaturing conditions andvisualized by Coomassie stain. The results are shown in FIG. 13A(non-reduced) and FIG. 13B (reduced).

C) Thermal Stability Measurement by DSF

The temperature at which a TFcA or TFc unfolds is determined byDifferential Scanning Fluorimetry (DSF) essentially as follows. The DSFassay is performed in the IQ5 Real Time Detection System (Bio-Rad). 20μl solutions of 15 uM TFcA or TFc, 1× Sypro Orange (Invitrogen LifeTechnologies), and 1×PBS are added to the wells of a 96 well plate. Theplate is heated from 20° C. to 90° C. with a heating rate of 1° C./minData is transferred to GraphPad Prism for analysis. Exemplary resultsfrom thermal stability determination by DSF for glycosylation sitemutants are shown in Table 25.

TABLE 25 DSF of Tandem Fcs protein Tm glyco_wt 60.5 glyco_1 55.5 glyco_257.9 glyco_3 57.9 glyco_4 57.3 glyco_5 56.3 glyco_6 51.5

Example 8 DSF Analysis of Backbone Variants Thermal StabilityMeasurement by DSF

The temperature at which a TFcA or TFc unfolds is determined byDifferential Scanning

Fluorimetry (DSF) as described above. Results are shown in Table 26below. These data show that backbone modifications such as the additionof a disulfide bridge and glycosylation mutations can improve thermalstability.

TABLE 26 DSF of Tandem Fcs with backbone variations Protein TmOnartuzumab 57.1 Onartuzumab-23 46.9 Onartuzumab-39 51.2 Onartuzumab-23E57.8 Onartuzumab-39Egy4 60.3 Onartuzumab-39Egy4-cetuximab 57.3Onartuzumab-39Egy4-panitumumab n/a Onartuzumab-39Egy4-2224 55.7

Example 9 Production and Analysis of Monovalent and Bispecific TFcMolecules Using Onartuzumab Binding Site Percent Monomer DeterminationUsing Size Exclusion Chromatography

50 μg of sample is injected on a TSKgel SuperSW3000 column (4.6 mm ID×30cm) using 20 mM sodium phosphate (+300 mM NaCl) as running buffer. Allmeasurements are performed on Agilent 1100 HPLC which is equipped withan auto sampler, a binary pump and a diode array detector. Percentmonomers are determined by analyzing the data in Chemstation software.Typically, all the samples are only protein A purified and at aconcentration of 5 mg/mL in 1×PBS.

TABLE 27 SEC Stability of TFcBA molecules at 4° C. Percent Percentmonomers monomers Molecule name (Day 0) (Day 7) Onartuzumab-23 72 76Onartuzumab-39 70 76 Onartuzumab-23E 89 89 Onartuzumab-39EGY4 81 81Onartuzumab-39EGY4-2224 88 88 Onartuzumab-39EGY4-panitumumab 85 85Onartuzumab-39EGY4-cetuximab 82 83

Fortebio Binding Protocol

Materials Required:

96-well, black, round, flat bottom, polypropylene microplates (GreinerBio-one #655209). Octet instrument and software (version 3.0). Protein Asensor tips (Fortebio, #18-5010). 1×PBS, antigen (his tagged cMET),antibodies.

Protocol:

All reagents are equilibrated and samples are brought to roomtemperature. Protein A sensor tips (Fortebio, #18-5010) are hydrated for10 min in 1×PBS. Kinetic assays are run using the Octet software andprocedure per manufacturer's instruction. Assay steps typically include:1-2 min of equilibration in 1×PBS, 4 min of antibody loading (conc: 50μg/mL in 1×PBS), 1-2 min of baseline stabilization, 4 min ofantibody:antigen association, and 4 min of antibody:antigendissociation. 1×PBS is used as the matrix throughout. Data are analyzedwith Octet Data Analysis software, processed, and fit to the curve using1:1 binding model to determine kinetic parameters (K_(d), K_(on) andK_(off))

TABLE 28 K_(d) of anti-cMet TFcs with various backbone modificationsMolecule or K_(d) determined by Fortebio antibody name (Binding tocMET.his) Onartuzumab-23E  1.3 nM Onartuzumab-39EGY4 1.25 nMOnartuzumab-23 1.24 nM Onartuzumab-39 1.26 nM Onartuzumab-39EGY4-2241.29 nM Onartuzumab-39EGY4-cetuximab  0.9 nMOnartuzumab-39EGY4-panitumumab  1.6 nM Onartuzumab  1.2 nM

Example 10 Signaling Inhibition by Onartuzumab and Bispecific Variants

To test the ability of the constructs in Table 28 to inhibit pMet, theTFc variants were tested in HGF-induced SW620 cells (ATCC cat #:CCL-227) as follows: On day 1, actively mid-log (about 80% confluence)growing cells (e.g., SW620 cells) are split in RPMI (+10%FBS+L/glutamine (2 mM)+Pen/Strep) media. Approximately 20,000 cells areseeded/well in a 96-well plate. On day 2, the media is changed from 10%FBS to serum-free media−RPMI+0.5% FBS (+L/glutamine+Pen/Strep) media. Onday 3, the HGF (stimulated control) and various inhibitors/antibodiesare diluted into the appropriate volume of serum free media. 100 μL ofeach inhibitor per concentration is added/well. The inhibitor is allowedto incubate at 37° C. for 2 hours. The cells are then washed 2× withcold PBS and later lysed in 50 μL/well of MPER (cat #PI78505, VWRInternational)+150 mM NaCl+Protease and Phosphatase Inhibitor buffer(cOmplete Protease Inhibitor Cocktail Tablet provided in EASY packs, cat#4693124001, Roche Diagnostics Corp; PhosSTOP Phosphatase InhibitorCocktail Tablets, cat #4906837001, Roche Diagnostics Corp). The lysatesare placed in −80° C. usually within 5 minutes after lysis.

For measurement of pMet signal, an ELISA kit was used (Human Phospho-HGFR/c-MET DuoSet IC Economy Pack, cat #DYC2480E, R&D Systems). A 384-wellHigh Binding Black Solid plate from Corning is coated with captureanti-MET antibody from R&D Systems at a final concentration of 4μg/mL/well in PBS buffer. The plates are left at overnight at roomtemperature. On day 4, the −80° C. lysate is thawed at room temperature.Plates are then washed 3 times with 50 μl/well in the BIOTEK platewasher with PBST (PBS with 0.05% Tween-20). The 384-well plates areblocked with 50 μL/well 2% BSA/PBS for 1 hour at room temperature.Duplicate lysates are pooled into one well and diluted 2-fold in 2%BSA/0.1% Tween-20/25% MPER/PBS. Recombinant standard curves are preparedby making 10×2-fold serial dilutions in 2% BSA/0.1% Tween-20/25%MPER/PBS. ELISA plates are washed with 0.05% Tween-20/PBS. 20 μL lysatesare transferred from the 96-well plate in quadruplicate to the 384-wellplate. Plates are incubated at room temperature for 2 hours and washed 3times with 0.05% Tween-20/PBS. 20 μL primary detectionanti-phosphotyrosine antibody, 4G10 (cat#05-321, Millipore/Upstate), isadded at a dilution of 1:1000 to the ELISA plates and incubated for 1hour at room temperature. 20 μL of SuperSignal ELISA PicoChemiluminescent Substrate (cat #PI37069, VWR International) is addedper manufacturer's directions and read on Envision Plate Reader (PerkinElmer). For data analyses, duplicate samples are averaged and error barsare used to represent the standard deviation between the two replicates.Inhibition curves and corresponding IC50 values are calculated usingGraphPad Prism software (GraphPad Software, Inc.) via regression of thedata to a 4 parameter logistic equation.

As shown in FIG. 16, all molecules tested inhibited pMet signaling in asimilar manner, without regard to the identity of the TFc core region.The bivalent onartuzumab_39 Egy4_2224, onartuzumab_39 Egy4_panitumumab,and onartuzumab_39 Egy4_cetuximab variants inhibited to a similar extentas did the monovalent onartuzumab_39 Egy4 variant.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain andimplement using no more than routine experimentation, many equivalentsof the specific embodiments described herein. Such equivalents areintended to be encompassed by the following claims. Any combinations ofthe embodiments disclosed in the dependent claims are contemplated to bewithin the scope of the disclosure.

INCORPORATION BY REFERENCE

The disclosure of each and every U.S. and foreign patent and pendingpatent application and publication referred to herein is specificallyincorporated by reference herein in its entirety.

Further Examples Section A-1

The following examples should not be construed as limiting the scope ofthis disclosure.

Throughout the examples, the following materials and methods are usedunless otherwise stated. In general, the practice of the techniques ofthe present disclosure employs, unless otherwise indicated, conventionaltechniques of chemistry, molecular biology, recombinant DNA technology,immunology (especially, e.g., antibody technology), pharmacology,pharmacy, and standard techniques in polypeptide preparation.

As used in these Examples and in the Figures, “HGF” refers to thehepatocyte growth factor (sometimes referred to as scatter factor),e.g., Preprotech, catalog #100-39.

The human cell lines used in the techniques and examples described belowmay be obtained, as indicated, from American Type Culture Collection(ATCC, Manassas, Va.). Cell lines used in the examples are: A549 cells(ATCC CCL-185); NCI-H441 (ATCC HTB-174); HCC827 (ATCC CRL-2868);NCI-H2170 (ATCC CRL-5928); and U-87 MG (ATCC HTB-14).

The humanized, anti-human-c-Met monoclonal antibody OA-5D5, as disclosedin U.S. Pat. No. 8,361,744 with SEQ ID NO:45 (heavy chain) and SEQ IDNO:46 (light chain), and from U.S. Patent Publication No. 20110300146with SEQ ID NO:196 (knob) is used as an anti-c-Met antibody control. Insome embodiments, the anti-c-Met heavy chain and knob sequences eachhave a single mutation SEQ ID NO:45 and SEQ ID NO:196 to remove the CH2glycosylation site. For example, see U.S. Pat. No. 7,476,724.

Antibodies described in this disclosure are abbreviated with thefollowing style: e.g. Antibody #1 is Ab#1, Antibody #2 is Ab#2, Antibody#13 is Ab#13, etc.

Example A-1 Tandem-Fc Bispecific Antibody Format

The general structure of the bispecific antibodies disclosed hereincomprises a bivalent antibody with a single Fab directed against c-Met,a “tandem-Fc” (TFc) backbone structure (described in copending PCTApplication Serial No. PCT/US2012/52490, e.g., SEQ ID NO:394 and 395),and a single scFv antibody fragment directed against EpCAM. The heavychain of the bispecific antibody comprises a single polypeptidecontaining the following domains (N-terminus to C-terminus):

(i) Heavy chain of the c-Met Fab

(ii) Hybrid hinge containing IgG1 upper hinge and IgG4 middle and lowerhinge

(iii) IgG4 CH2 domain #1 with T299K mutation

(iv) IgG1 CH3 domain #1 with T366S/L368A/Y407V mutations

(v) Disulfide bridge motif #1 (KSCDKT)

(vi) (G₄S)₈ polypeptide linker

(vii) Middle and lower hinge of IgG4

(viii) IgG4 CH2 domain #2 with T299D mutation

(ix) IgG1 CH3 domain #2 with T366W mutation

(x) Disulfide bridge motif #2 (GEC)

(xi) Connecting polypeptide linker

(xii) Anti-EpCAM scFv

A schematic of the antibody domain structure can be found in FIG. 18.

Example A-2 Humanization of Anti-EpCAM scFv

A structure-guided approach was employed to create a panel of 13humanized anti-EpCAM scFv variants deriving from the murine parentsequence (SEQ ID NOs: 486 & 487). The initial homology models of scFvswere built by MolIDE using independent zero-gap templates for VH and VLdomains and energy-refined using SCWRL A review of MolIDE and SCWRL isdisclosed, e.g., in Nature Protocols 3:1832-1847 (2008). The potentialimpact of the candidate mutations on stability was conducted by visualinspection using PyMOL or calculating energy differences using Eris.These scFv variants were incorporated into 13 bispecific antibodies(Ab#1, Ab#2, Ab#3, Ab#4, Ab#5, Ab#6, Ab#7, Ab#8, Ab#9, Ab#10, Ab#11,Ab#12, and Ab#13). Permutations used include three different antibodyframeworks, removal of deamidation or oxidation sites in the CDRsequences, VH/VL sequence orientation, disulfide bridge addition, andintra-scFv protein linker sequence. An overview of the designpermutations tested is shown in FIG. 2.

Example A-3 Protein Expression and Purification of Antibodies

A) Transient Transfection

Antibodies are expressed using a transient transfection protocolutilizing Freestyle 293F cells (Invitrogen). The nucleic acids encodingthe proteins are cloned as single proteins into expression plasmidsusing standard recombinant DNA techniques. An exemplary expressionvector employed is pCEP4 (Life Technologies, catalog #R790-07). Heavychain and light chain sequences for the antibodies are cloned intoseparate vectors. Expression plasmids are transfected using polyethyleneimine (2.5 μg per ml of cell culture) and DNA (1:1 ratio of heavy chainand light chain, with a combined total of 1 μg DNA per ml of cellculture). Transfected cells are incubated at 37° C., 5% CO₂ for six daysand then harvested by centrifugation at 6000 g for 30 minutes followedby 0.2 μM filtration of the supernatant.

B) Stable transfection

Antibodies are expressed via stable transfection into suspension adaptedCKO-K1 cells (Chinese hamster ovary; ATCC catalog #CCL-61) essentiallyas follows. The nucleic acids encoding the antibodies are cloned assingle proteins into expression plasmids using standard recombinant DNAtechniques. An exemplary expression vector employed is pMP 10K(SELEXIS). Heavy chain and light chain sequences for the antibodies arecloned into separate vectors. Heavy and light chain expression plasmidsare linearized, purified using QIAquick purification kit (QIAGEN), andco-transfected at a 1:1 ratio as follows.

Suspension-adapted CHO-K1 cells are grown in Hyclone SFM4CHO media(Fisher Scientific, catalog #SH30548.02) supplemented with 8 mML-glutamine (Life Technologies, catalog #25030-081) to a density of 2million cells per ml. On the day of transfection, the cells areresuspended in Opti-MEM serum free media (Gibco, catalog #31985-062) ata density of 80,000 cells per ml. The cells (500 μL) are thentransfected with 1 μg of total linearized DNA (including 10 ng of pNeovector, an in-house vector carrying the geneticin selection marker)using 2.75 μL of Lipofectamine (Life Technologies, catalog #15338-100)in a 24 well plate. After 3 hours, 1 ml of recovery media (HAMS-F12(Gibco)+10% FBS) is added, and the transfected cells allowed to recoverfor 48 hours. The cells are then expanded into a 96-well plate, and theselection marker geneticin was added to the recovery media at 500 μg/ml.After 4 more days, the media is replaced with serum free Hyclone SFM4CHOmedia (supplemented with L-glutamine), and the transfected cells allowedto adapt. After a week, the selected cells form colonies, and the wellscontaining single colonies are tested for desired characteristics withwestern blots from the supernatant. The desired clones are expanded to a24 well plate, then to a T-25 flask, and eventually to a 125 ml shakeflask. Expression of antibody by the desired clones are confirmed withSDS-PAGE, and scaled up to the desired volume. The cells are harvestedby centrifugation (6000 g, 30 min) when the viability falls below 80%,and the supernatant filtered using a 0.22 μm filter. Cell viability isassessed using trypan blue exclusion assay and is measured automaticallyusing a Vi-CELL Cell Viability Analyzer (Beckmann Coulter).

C) Purification

All proteins are purified using a protein A affinity chromatographyprotocol in accordance with manufacturer's instructions, using MabSelect(GE Healthcare) as the Protein A affinity resin, on a AKTA Explorer 100FPLC (GE Healthcare). The protein A affinity step is used to selectivelyand efficiently bind the antibodies out of harvested cell culturefluids. This removes >95% of product impurities in a single step withhigh yields and high throughput. The purified material is concentratedusing Vivaspin centrifugal concentrators (GE Healthcare), and dialyzedinto PBS using Slide-A-Lyzer G2 Dialysis Cassettes (Pierce), bothaccording to manufacturer's instructions.

Example A-4 Biophysical Characterization of Expressed Antibodies

A) SDS-PAGE Analysis

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) isused to assess the molecular weight of expressed antibodies, anddetermine whether an antibody is properly formed or assembled.Antibodies (2 μg each) are run on a 4-12% SDS-PAGE gel in eithernon-reducing or reducing conditions and visualized by Coomassiebrilliant blue stain.

OA-5D5 was expressed using stable transfection and purified as describedabove, and visualized using SDS-PAGE. FIG. 3 depicts results for OA-5D5deriving from 5 different clones under reducing and non-reducingconditions. Under non-reducing conditions, all OA-5D5 clones wereobserved to have a band with molecular weight of approximately 100kilodaltons, corresponding to properly associated monovalent antibody.OA-5D5 clones also showed a band at approximately 25 kilodaltons, likelycorresponding to excess light chain or Fc domain Under reducingconditions, all OA-5D5 clones demonstrated two bands of approximately 25and 50 kilodaltons, likely corresponding to OA-5D5 light chain and Fcdomain (25 kDa) and heavy chain (50 kDa).

Ab#5, Ab#7, and Ab#13 were expressed using stable transfection andpurified as described above, and visualized using SDS-PAGE. FIG. 4depicts results for Ab#5, Ab#7, and Ab#13 under reducing andnon-reducing conditions. For both conditions, Ab#5, Ab#7, and Ab#13 wereobserved to run at the expected molecular weight. Under non-reducingconditions, Ab#5, Ab#7, and Ab#13 were observed to have largely a singleband with molecular weight of approximately 125 kilodaltons,corresponding to properly associated antibody light and heavy chain.Under non-reducing conditions, two bands of approximately 25 and 100kilodaltons were observed corresponding to antibody light and heavychain.

B) Percent Monomer Determination Using Size Exclusion Chromatography

Size exclusion chromatography may be used to assess the purity ofexpressed antibodies by characterizing the presence of aggregates orantibody fragments. To assess the percent monomer of the antibodies, 50μg of sample is injected on a TSKgel SuperSW3000 column (4.6 mm ID×30cm; Tosoh Bioscience) using 20 mM sodium phosphate+300 mM sodiumchloride as running buffer. All measurements are performed on an Agilent1100 HPLC which is equipped with an auto sampler, a binary pump and adiode array detector. Percent monomers are determined by analyzing thedata in Agilent ChemStation software. Typically, samples are onlyprotein A purified and run at a concentration of 5 mg/ml in 1×PBS.

Ab#1, Ab#2, Ab#3, Ab#4, Ab#5, Ab#6, Ab#7, Ab#8, Ab#9, Ab#10, Ab#11,Ab#12, and Ab#13 were expressed using transient transfection andpurified as described above, and OA-5D5 was expressed using stabletransfection and purified as described above. All molecules wereanalyzed via size exclusion chromatography. Data on approximate percentmonomeric character for the Protein A purified antibodies are shownbelow in Table 1.

TABLE A-1 Monomer Antibody percentage Ab#1 74.2 Ab#2 75.1 Ab#3 74.2 Ab#487.5 Ab#5 86.5 Ab#6 86.1 Ab#7 87.1 Ab#8 86.3 Ab#9 95.1 Ab#10 91.5 Ab#1190.4 Ab#12 90.9 Ab#13 92.2 OA-5D5#1 78 OA-5D5#2 75 OA-5D5#3 24 OA-5D5#489 OA-5D5#5 98

Example 5 Binding Affinity of Antibodies to Recombinant c-Met and EpCAM

Kinetic assays to measure the association rate, dissociation rate, andbinding affinity of antibodies for, e.g., the molecular targets c-Metand EpCAM may be measured using the Octet platform (ForteBio) permanufacturer's instructions. Materials required are as follows:

-   -   96-well black flat bottom polypropylene microplates (Greiner        Bio-one #655209)    -   Octet instrument and software (version 3.0)    -   Protein A sensor tips (ForteBio, #18-5010)    -   1×PBS    -   Recombinant polyhistidine-tagged c-Met    -   Recombinant polyhistidine-tagged EpCAM-Fc

For Octet analysis, all reagents and samples are brought to roomtemperature. Protein A sensor tips (ForteBio, #18-5010) are hydrated for10 minutes in 1×PBS. Assay steps include: 1-2 minutes of equilibrationin 1×PBS, 4 minutes of antibody loading (concentration of 50 μg/ml in1×PBS), 1-2 minutes of baseline stabilization, 4 minutes ofantibody:antigen association, and 4 minutes of antibody:antigendissociation. 1×PBS is used as the matrix throughout. Data are analyzedwith Octet Data Analysis software, processed, and fit to a 1:1 bindingmodel to determine kinetic parameters (K_(d), k_(on) and k_(diss)).

Table A-2 describes approximate EpCAM binding affinity and dissociationrate as determined for Ab#1, Ab#2, Ab#3, Ab#4, Ab#5, Ab#6, Ab#7, Ab#8,Ab#9, Ab#10, Ab#11, Ab#12, and Ab#13.

Table A-3 describes a repeat analysis for Ab#5, Ab#7, and Ab#13. For therepeat experiment, K_(d) and k_(diss) values for Ab#5, Ab#7, and Ab#13are substantially equivalent to those determined for Ab#5, Ab#7, andAb#13 in the initial experiment.

TABLE A-2 Antibody K_(d) (M) k_(diss) (1/s) Ab#1 1.37E−08 1.21E−03 Ab#2no binding no binding Ab#3 1.70E−08 1.74E−03 Ab#4 1.94E−08 1.74E−03 Ab#53.57E−09 3.46E−04 Ab#6 1.12E−08 1.22E−03 Ab#7 2.59E−08 1.32E−03 Ab#81.67E−08 9.80E−04 Ab#9 1.00E−08 4.30E−04 Ab#10 2.62E−08 2.11E−03 Ab#114.57E−09 4.09E−04 Ab#12 2.45E−08 1.66E−03 Ab#13 4.98E−09 3.60E−04

TABLE A-3 Antibody K_(d) (M) k_(diss) (1/s) Ab#5 2.67E−09 1.94E−04 Ab#71.54E−08 1.37E−03 Ab#13 4.69E−09 3.23E−04

Table A-4 describes approximate c-Met binding affinity and dissociationrate as determined for Ab#5 and Ab#7.

TABLE A-4 Antibody K_(d) (M) k_(diss) (1/s) Ab#5 3.40E−09 5.88E−04 Ab#72.81E−09 5.54E−04

Example A-6 Plate-Based Bispecific Antibody Binding Assay

The antibodies disclosed herein are designed to simultaneously bind bothEpCAM and c-Met. A plate-based sandwich-type assay may be used todemonstrate the co-binding of the antibodies. The assay is performed asfollows. Reacti-Bind 96-well plates (Pierce, catalog #15041) are coatedwith 50 μL of 2 μg/ml cMet-Fc (Merrimack Pharmaceuticals) in PBS, andincubated overnight at 4° C. On the next day, the plates are washed withPBS-T (PBS+0.05% Tween-20), blocked for 1 hour at room temperature with100 μL of Protein-Free Blocking Buffer (Pierce, catalog #37572), andwashed again with PBS-T. Plates are next incubated for 2 hours at roomtemperature with 100 μL of antibody in PBS-T+10% FBS (highest antibodyconcentration of 500 nM, with 10 subsequent three-fold dilutions, andone blank well for background subtraction). The plates are washed withPBS-T, and 50 μl of 1 μg/ml EpCAM-Fc-His (Merrimack Pharmaceuticals) inPBS-T is added for 1 hour at room temperature. Plates are washed withPBS-T and 50 μl of anti-His-HRP (Abcam, catalog #ab1187; 1:20,000 inPBS-T) is added and incubated (covered) for 1 hour at room temperature.Plates are again washed with PBS-T, followed by addition of 100 μl of3,3′,5,5′-tetramethylbenzidine (TMB; Cell Signaling, catalog #7004) andincubation between 5-15 minutes at room temperature. The substratereaction is stopped with 100 μl of Stop Solution (Cell Signaling,catalog #7002). The well absorbance is measured at 450 nm using anEnvision plate reader (PerkinElmer), and the resulting data analyzed andplotted using GraphPad Prism (GraphPad Software, Inc.).

Using the above methods, OA-5D5, Ab#5, Ab#7, and Ab#13 were assessed forthe ability to simultaneously bind both c-Met and EpCAM. Representativeresults of the assay are shown in FIG. 22. The three bispecificantibodies tested both exhibited a dose-dependent increase inabsorbance, confirming ability to bind both recombinant c-Met and EpCAM,whereas monospecific control antibody showed no significant signal inthe assay.

Example A-7 Quantitative Flow Cytometry Assessment for Cell SurfaceExpression of c-Met and EpCAM

For measurements of cell surface expression of, e.g., cMet and EpCAMlevels, quantitative flow cytometry is performed using the QuantumSimply Cellular kit (Bangs Laboratories).

Cells are grown in exponential phase using standard cell culture mediacontaining 10% FBS, and are passaged at least twice before the start ofthe experiment. On the day of the experiment, cells are visuallyassessed under a microscope to confirm between 60% and 80% confluence.Cells are detached from the culture plate by addition of 20 μl trypsin,and once a majority of cells are detached (as assessed visually bymicroscope) the trypsin is inactivated using cell culture mediacontaining 10% FBS. The cells are centrifuged at 500 g, resuspended inflow cytometry buffer (2% FBS+0.1% sodium azide in PBS), and seeded at adensity of 50,000 cells per well in a 96-well plate (BD Biosciences,catalog #62406-015).

In a separate 96-well plate, 2 drops of Quantum Simply Cellularanti-mouse IgG coated beads (Bangs Laboratories, catalog #815) oranti-human IgG coated beads (Bangs Laboratories, catalog #816) are addedper well. Each bead kit contains 5 bead populations (1 blank and 4 beadswith increasing levels of Fc-specific capture antibody). Each coatedpopulation binds a specific number of monoclonal antibodies of theappropriate species (the “ABC” value), and thus serves as a standardcurve for quantification when beads are labeled to saturation with thesame monoclonal antibody that is used to label cell surface protein.

Anti-cMet human antibody (h224G11-TH7 clone) or anti-EpCAM murineantibody (BD Biosciences, catalog #347200) conjugated with APC are addedto the cells and the beads (200 nM antibody concentration in 80 μl offlow cytometry buffer). Antibody is allowed to incubate for 30 minutesat 4° C. The plates are centrifuged and washed twice with 100 μL ofice-cold flow cytometry buffer. After the last wash, the cells and beadsare centrifuged and resuspended in 100 μL of ice-cold flow cytometrybuffer and read using the appropriate fluorescence filter on a flowcytometer (BD FACSCanto). Channel values for the bead populations arerecorded in the bead lot-specific QuickCal template provided in theQuantum Simply Cellular kit. A regression is performed that relatesfluorescence signal to the beads' ABC values. ABC values are assigned tostained cell samples using this standard curve. If monovalentantibody-to-cell surface receptor binding is presumed, then the ABCvalue equals the number of surface receptors.

Table 5 lists EpCAM and c-Met cell surface expression levels in celllines measured using the above protocol. U-87 MG and A549 cells have lowEpCAM expression level, which NCI-H441, HCC827, and NCI-H2170 cells havehigh levels of EpCAM expression.

TABLE A-5 c-Met EpCAM EpCAM:c-Met Cell line (#/cell) (#/cell) ratio U-87MG 3.2 × 10⁴ 4.0 × 10² 1.3 × 10² A549 7.2 × 10⁴ 2.4 × 10⁴ 3.3 × 10⁻¹NCI-H441 3.2 × 10⁵ 2.5 × 10⁶ 7.8 × 10⁰ HCC827 2.1 × 10⁵ 2.1 × 10⁶ 1.0 ×10¹ NCI-H2170 5.8 × 10⁴ 3.9 × 10⁶ 6.7 × 10¹

Example A-8 Assessment of Basal c-Met Pathway Activation

A) Antibody-Induced Activation of c-Met Mediated Cell Signaling

Activation of c-Met pathway signaling is an observable characteristic ofbivalent antibodies against c-Met. The ability of the antibodies toactivate c-Met mediated signal transduction can be assessed using aphospho-c-Met (“pMet”) enzyme-linked immunosorbent assay (ELISA),essentially as follows.

On Day 1 of the experiment, A549 cells actively growing in mid-log phase(60-80% confluence) are plated at approximately 20,000 cells per well ina 96-well plate using RPMI media containing 10% FBS and supplementedwith penicillin/streptomycin and 2 mM 1-glutamine. On Day 2, the mediais aspirated and replaced with low-serum RPMI media containing 0.5% FBSand supplemented with penicillin/streptomycin and 1-glutamine, and thecells incubated overnight. On Day 3, dilution series of both HGF(serving as positive control, with starting concentration of 40 nMdiluted serially 3-fold) and antibodies (with starting concentration of300 nM diluted serially 3-fold) are prepared using low-serum media. 100μL from each dilution series concentration is then added to a platewell, and the cells are allowed to incubate at 37° C. for 10 minutes.Cells are then washed 2× with cold PBS and lysed in 50 μL per well ofM-PER solution (catalog #PI78505, VWR International)+150 mMNaCl+protease and phosphatase inhibitor (cOmplete Protease InhibitorCocktail Tablet provided in EASY packs, catalog #4693124001, RocheDiagnostics Corp; PhosSTOP Phosphatase Inhibitor Cocktail Tablets,catalog #4906837001, Roche Diagnostics Corp). Cell lysates are stored at−80° C. within approximately 5 minutes after lysis.

For measurement of pMet signal, an ELISA kit is used (Human Phospho-HGFR/c-MET DuoSet IC Economy Pack, catalog #DYC2480E, R&D Systems). On Day3, a 384-well High Binding Black Solid plate (Corning) is coated withcapture anti-c-Met antibody at a final concentration of 4 μg per ml in20 μl per well of PBS. The plates are left at overnight at roomtemperature. On Day 4, the −80° C. lysate is thawed at room temperature.The 384-well plates are blocked with 50 μL per well of a solution of 2%BSA in PBS for 1 hour at room temperature. Duplicate lysates are pooledinto one well and diluted 2-fold in a solution of 2% BSA/0.1%Tween-20/25% M-PER/PBS. A standard curve of recombinant pMet is preparedby making a series of ten 2-fold serial dilutions (starting at 40 nMconcentration) in 2% BSA/0.1% Tween-20/25% M-PER/PBS. Wells containingbuffer only are used as control for background signal on the plate.ELISA plates are washed with a solution of 0.05% Tween-20 in PBS, and 20μL of lysate or recombinant standard curve is transferred from the96-well plate in duplicate to the 384-well plate. Plates are incubatedat room temperature for 2 hours and washed with a solution of 0.05%Tween-20 in PBS. Next, 20 μL of primary detection antibody conjugatedwith horseradish peroxidase is added per well to the ELISA plates andincubated for 2 hours at room temperature. SuperSignal ELISA PicoChemiluminescent Substrate (catalog #PI37069, VWR International) isadded per manufacturer's directions and read on an Envision Plate Reader(Perkin Elmer). For data analyses, the duplicate samples are averagedand error bars are used to represent the standard deviation between thetwo replicates. Data are background-subtracted and regressed against therecombinant standard curve. Subsequently, Met stimulation curves andcorresponding EC50 values are calculated using GraphPad Prism software(GraphPad Software, Inc.) via regression of the data to a 4-parameterlogistic equation.

Using the above protocol, antibody-induced activation of Met to yieldphospho-Met (pMet) was tested for a bivalent IgG. The result of thisexperiment is shown in FIG. 6. Treatment with HGF, as expected, produceda strong pMet signal. Bivalent IgG treatment also caused c-Metphosphorylation, to a level roughly one-half that of the native c-Metligand HGF. In contrast, monovalent antibody OA-5D5 did not cause c-Metphosphorylation.

Ab#5, Ab#7, Ab#13, and OA-5D5 were subsequently tested in the aboveassay for their ability to cause c-Met phosphorylation. The results ofthis experiment are shown in FIG. 7. Again, treatment of HGF resulted ina strong pMet dose-response signal, while monovalent antibody OA-5D5 didnot cause c-Met phosphorylation. Treatment with the bispecificantibodies did not result in c-Met phosphorylation.

B) Antibody-Induced Activation of Cell Proliferation

Antibodies targeting the c-Met receptor often have the ability tostimulate DNA synthesis and cell proliferation, which is detrimental forthe development of a drug targeting human malignancies. This propertycan be assessed as follows.

For determination of the ability of the antibodies to induce cellproliferation, cells are first plated in a 384-well plate using 50 μl ofRPMI Media 1640 (Gibco) containing 0.5% FBS. A549 cells are plated at800 cells per well, NCI-H441 cells are plated at 2500 cells per well,and HCC827 cells are plated at 1500 cells per well. After overnightincubation of the plate, media is aspirated and replaced with 50 μl ofRPMI Media 1640 containing 0.5% FBS and either 2.5 nM HGF alone as apositive control, or 500 nM of the antibody of interest. The plate isthen incubated at 37° C., and cell growth monitored using an Incucytereal-time imaging system (Essen Bioscience). Images of the plate wellsare recorded using phase contrast microscopy every six hours for fourdays, and cell confluence (reported as a percentage) calculatedaccording to manufacturer's software. Confluence data is exported intoGraphPad Prism software (GraphPad Software, Inc.) for plotting andvisualization.

Using the above experimental protocol, Ab#5, Ab#7, and Ab#13 were testedin A549, NCI-H441, and HCC827 cells. Data from these experiments areplotted in FIG. 8A (A549 cells), FIG. 8B (NCI-H441 cells), and FIG. 8C(HCC827 cells). Each data point shown represents the mean confluence ofcells in four replicate plate wells. For all three cell lines, HGFstimulation resulted in increased confluence of cells relative to thelow serum control, corresponding to an increase in cell proliferation.In NCI-H441 and HCC827 cells, none of the tested bispecific antibodiesresulted in greater cell confluence than the low serum control. In A549cells, Ab#7 and Ab#13 showed no increase of cell confluence above thelow serum control, while Ab#5 modestly increased cell confluence abovethe low serum control.

Example A-9 Assessment of Inhibition of HGF-Induced c-Met PathwayActivation

A) Antibody-Mediated Inhibition of HGF-Induced Cell Signaling

The ability of antibodies to inhibit HGF-induced, c-Met mediated signaltransduction may be assessed in A549 and NCI-H2170 cells using aphospho-AKT (“pAKT”) ELISA.

On Day 1 of the experiment, cells actively growing in mid-log phase(60-80% confluence) are plated at approximately 20,000 cells per well ina 96-well plate using RPMI media (+10% FBS+2 mM L-glutamine+Pen/Strep).On Day 2, the media is aspirated and replaced with low-serum RPMI media(+0.5% FBS+L-glutamine+Pen/Strep), and the cells incubated overnight. OnDay 3, dilution series of antibodies (with starting concentration of 300nM diluted serially 3-fold) are prepared using low-serum media. 100 μLfrom each dilution series concentration is then added to a plate well,and the cells are allowed to incubate at 37° C. for 2 hours. Next, theantibody containing media is aspirated and replaced with 100 μL ofantibody containing media additionally containing 1 nM HGF. The cellsare allowed to incubate with the HGF containing media at 37° C. for 10minutes. Cells are then washed 2× with cold PBS and lysed in 50 μL perwell of M-PER solution (catalog #PI78505, VWR International)+150 mMNaCl+protease and phosphatase inhibitor (cOmplete Protease InhibitorCocktail Tablet provided in EASY packs, catalog #4693124001, RocheDiagnostics Corp; PhosSTOP Phosphatase Inhibitor Cocktail Tablets,catalog #4906837001, Roche Diagnostics Corp). Cell lysates are stored at−80° C. within approximately 5 minutes after lysis.

For measurement of pAKT signal, an ELISA assay was used. A 384-well HighBinding Black Solid plate (Corning) is coated with capture anti-AKTantibody (Millipore, catalog #05-591M) at a final concentration of 4 μgper ml in 20 μl per well of PBS. The plates are left at overnight atroom temperature. On Day 4, the −80° C. lysate is thawed at roomtemperature. The 384-well plates are blocked with 50 μL per well of asolution of 2% BSA in PBS for 1 hour at room temperature. Duplicatelysates are pooled into one well and diluted 2-fold in a solution of 2%BSA/0.1% Tween-20/25% M-PER/PBS. A standard curve of recombinant pAKT(Millipore, catalog #14-276) is prepared by making a series of ten2-fold serial dilutions (starting at 60 nM concentration) in 2% BSA/0.1%Tween-20/25% M-PER/PBS. Wells containing buffer only are used as controlfor background signal on the plate. ELISA plates are washed with asolution of 0.05% Tween-20 in PBS, and 20 μL of lysate or recombinantstandard curve is transferred from the 96-well plate in duplicate to the384-well plate. Plates are incubated at room temperature for 2 hours andwashed with a solution of 0.05% Tween-20 in PBS. Next, 20 μL of 1:1000diluted biotinylated secondary antibody (Cell Signaling Technology,catalog #5102) is added per well to the ELISA plates and incubated for 1hr at room temperature. ELISA plates are then washed with a solution of0.05% Tween-20 in PBS. 20 μL of diluted streptavidin conjugated with HRP(R&D Systems, catalog #DY998) is added to the ELISA plates and incubatedfor 30 minutes at room temperature. SuperSignal ELISA PicoChemiluminescent Substrate (catalog #PI37069, VWR International) isadded per manufacturer's directions and read on an Envision Plate Reader(Perkin Elmer). For data analyses, the duplicate samples are averagedand error bars are used to represent the standard deviation between thetwo replicates. Data are background-subtracted and regressed against therecombinant standard curve. Subsequently, pAKT inhibition curves andcorresponding IC₅₀ values are calculated using GraphPad Prism software(GraphPad Software, Inc.) via regression of the data to a 4 parameterlogistic equation.

Using the above protocol, the ability of Ab#5, Ab#7, Ab#13, and OA-5D5to inhibit HGF stimulated pAKT was tested in both A549 and NCI-H2170cells. Typical results of these experiments are shown in FIG. 9. In theA549 cells, which have low EpCAM expression, Ab#5, Ab#7, and Ab#13 hadsubstantially equivalent inhibition potency relative to OA-5D5. In theNCI-H2170 cells, which have high EpCAM expression, Ab#5, Ab#7, and Ab#13exhibited approximately a 10-fold to 20-fold improvement in pAKT IC50relative to OA-5D5.

B) Antibody-Mediated Inhibition of HGF-Induced Cell Proliferation

The ability of antibodies to inhibit HGF-induced, c-Met mediated cellproliferation may be assessed, for example, in U-87 MG, A549, andNCI-H441 cells as follows.

For determination of the ability of the antibodies to inhibitHGF-induced, c-Met mediated cell proliferation, cells are first platedin a 384-well plate using 50 μl of RPMI Media 1640 (Gibco) containing0.5% FBS. A549 cells are plated at 800 cells per well, U-87 MG cells areplated at 1500 cells per well, and NCI-H441 cells are plated at 3000cells per well.

For A549 and NCI-H441 cells, after overnight incubation of the plate,media is aspirated and replaced with 50 μl of RPMI Media 1640 containing0.5% FBS only (negative control), 0.5% FBS plus 0.625 nM HGF (positivecontrol), or 0.5% FBS plus 0.625 nM HGF and 500 nM of the antibody ofinterest.

For U-87 MG cells, which secrete autocrine HGF, after overnightincubation of the plate, media is aspirated and replaced with 50 μl ofRPMI Media 1640 containing either 0.5% FBS only (negative control) or0.5% FBS plus 500 nM of the antibody of interest.

The plate is then incubated at 37° C., and cell growth monitored usingan Incucyte real-time imaging system (Essen Bioscience). Images of theplate wells are recorded using phase contrast microscopy every six hoursfor four days, and cell confluence (reported as a percentage) calculatedaccording to manufacturer's software. Confluence data is exported intoGraphPad Prism software (GraphPad Software, Inc.) for plotting andvisualization.

Using the above experimental protocol, Ab#5 and OA-5D5 were tested usingU-87 MG and NCI-H441 cells. Results from a representative experiment areplotted in FIG. 10. Each data point shown represents the mean confluenceof cells in four replicate plate wells. In the U-87 MG cells, which havelow EpCAM expression, both Ab#5 and OA-5D5 were substantiallyequivalently able to decrease cell confluence. In the NCI-H441 cells,which have high EpCAM expression, Ab#5 but not OA-5D5 exhibited anability to decrease HGF-induced cell confluence below basal 0.5% serumconditions.

Using the above experimental protocol, Ab#7 and OA-5D5 were tested usingA549 and NCI-H441 cells. Results from a representative experiment areplotted in FIG. 11. Each data point shown represents the mean confluenceof cells in four replicate plate wells. In the A549 cells, which havelow EpCAM expression, both Ab#7 and OA-5D5 were able to inhibitHGF-induced cell confluence to the level of the 0.5% serum control. Inthe NCI-H441 cells, which have high EpCAM expression, Ab#5 decreasedcell confluence nearly to the basal 0.5% serum control, while OA-5D5showed a decreased ability to inhibit cell confluence.

Example A-10 Assessment of Antibody-Induced c-Met Degradation

To assess the ability of antibodies to induce degradation of c-Metreceptor, 7,000 A549 or 15,000 NCI-H2170 cells are plated into a 96-wellflat bottom plate using RPMI media containing 10% FBS and supplementedwith penicillin/streptomycin and 1-glutamine. The following day, mediais replaced with 100 μl of low-serum RPMI 1640 media (Gibco) containing0.5% FBS supplemented with penicillin/streptomycin and 1-glutamine.After overnight incubation, the low-serum media is aspirated andreplaced with low-serum media additionally containing the antibody ofinterest. Cells are incubated at 37° C., 5% CO2 for 24 hours. Upon thecompletion of the 24 hour incubation, the cells are washed with PBStwice and then lysed with M-PER (mammalian protein extraction reagent;Pierce, catalog #78505) containing protease and phosphatase inhibitors(cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail Tablet providedin EASY packs, catalog #04693159001, Roche Diagnostics Corp; PhosSTOPPhosphatase Inhibitor Cocktail Tablets, catalog #4906837001, RocheDiagnostics Corp).

Cell lysates are evaluated in duplicate by a total c-Met ELISA(Invitrogen, catalog #KH00251) according to the manufacturer'sinstructions. Signal was detected using an Envision Plate Reader (PerkinElmer). For each cell line, the total c-Met expression was normalized tothe amount detected in a no inhibitor media control to allow forcomparison across cell lines.

Using the above method, OA-5D5 and Ab#5 were tested for the ability todegrade c-Met receptor in A549 and H2170 cells. The resulting data isshown in FIG. 12. In the low EpCAM A549 cells, neither OA-5D5 nor Ab#5induced more than 20% c-Met degradation. In the high EpCAM NCI-H2170cells, OA-5D5 reduced c-Met expression by approximately 30%, while Ab#5reduced c-Met expression by approximately 65%.

Example A-11 Pharmacokinetic Properties of Antibodies in Mice

To determine the terminal elimination half-life of the antibodies, Nu/Numice (Charles River Laboratories) are dosed by intravenous bolus withantibody, and bleeds taken at 0.25, 1, 4, 8, 24, 48, 72, 96, 144, 192,240, and 288 hours. Three mice are bled per time point (using eithersaphenous vein or terminal bled) and the serum collected (between 10-200μl) from individual animals. For serum sample analysis, Reacti-Bind96-well plates (Pierce) are coated with 50 μl of 1 μg/ml of goat anti-Fcantibody (Abcam, catalog #ab98616) in PBS and incubated overnight at 4°C. On the next day, plates are washed with PBS-T (PBS+0.05% Tween-20),blocked for 1 hour at room temperature with 100 μl of Protein-FreeBlocking Buffer (Pierce, catalog #37572), and washed again with PBS-T.The plates are then incubated for 2 hours at room temperature with 100μL of samples and standard curves. For the standard curves, antibodiesof interest are diluted to an initial concentration of 12 μg/ml in PBS-Twith 10 additional 3-fold dilutions and a final well blank. Serumsamples are diluted at 1:50 in PBS-T with 10 additional 3-fold dilutionsand a final well blank. The plates are washed with PBS-T, and 100 μl ofanti-Fc-HRP antibody (Abcam, catalog #ab99759; 1:20,000 in PBS-T) isadded and incubated (covered) for 1 hour at room temperature. Plates areagain washed with PBS-T, followed by addition of 100 μl of3,3′,5,5′-tetramethylbenzidine (TMB; Cell Signaling, catalog #7004) andincubation between 5-15 minutes at room temperature. The substratereaction is stopped with 100 μl of Stop Solution (Cell Signaling,catalog #7002). The well absorbance is measured at 450 nm using anEnvision plate reader (PerkinElmer), and the back-calculated valuesgenerated using SoftMax Pro. Pharmacokinetic curve fitting is performedin MATLAB (The Mathworks) using non-linear regression analysis of atwo-compartment biexponential model:

Concentration=Ae ^(−αt) +Be ^(−β)

For the non-linear regression, data is assumed to follow a proportionalerror model, and thus for the regression the model and data are weightedusing the inverse of data magnitude. Terminal elimination half-life iscalculated as follows:

$t_{\frac{1}{2},{el}} = \frac{\ln \; (2)}{\beta}$

Ab#5, Ab#7, and Ab#13 were evaluated using the above methods, and theresulting mean and standard deviation (6 data points for each timepointrepresenting 3 mice and 2 well replicates per mouse) are plotted in FIG.13A (Ab#5), FIG. 13B (Ab#7), and FIG. 13C (Ab#13). Model fits to thedata are shown as a solid line. Terminal half-life data is given belowin Table 6.

TABLE A-6 Dose Terminal elimination Antibody (mg/kg) half-life (hr) Ab#530 60 Ab#7 30 121 Ab#13 30 64

Example A-12 Evaluation of Antibody Activity on U-87 MG Tumors Implantedinto Nude Mice

The in vivo activity of Ab#7 and OA-5D5 is evaluated as follows.Six-to-seven-week-old female Nu/Nu mice (Charles River Laboratories) areinjected subcutaneously with 5×10⁶ U-87 MG cells/mouse (ATCC) using aninjection volume of 200 μl PBS. Seven days post-injection, initial tumorvolumes are measured using the following formula: (π/6)*L*W². Mice aresegregated into groups of 10, and subsequently treated with PBS control,bispecific antibody, or OA-5D5 by intraperitoneal injection every 3days. The doses of the bispecific antibody correspond to approximately30 mg/kg based on body weight, while the doses of OA-5D5 dosing wereapproximately 24 mg/kg (an equal molar level). Tumor measurements aredetermined twice weekly throughout the study. Plotted data of tumor sizerepresents mean and standard error of the mean for each measurement.

Data from a representative experiment using the above protocol are shownin FIG. 31. Both Ab#7 and OA-5D5 were able to regress subcutaneouslyimplanted U-87 MG tumors to a similar extent.

Example A-13 Sequence Information for Additional Embodiments

Amino Acid (aa) and Nucleotide (nt) sequences

Underline: CDRs

Bold sequence: Stabilization mutations

>anti-c-Met-LC aa (SEQ ID NO: 400) (Light Chain for SEQ ID NOs: 403-415)DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISS                              CDR1                   CDR2LQPEDFATYYCLQANSFPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL              CDR3QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC** >RESERVED SEQUENCE(SEQ ID NO: 401)XXXX >anti-c-Met-LC nt (Light Chain for SEQ ID NOs: 403-415)(SEQ ID NO: 402)gacattcagatgacccagtctccttcaagcgtcagcgcttccgtgggcgaccgggtcaccatcacatgcagagcctcccaggggattagctcctggctggcttggtatcagcagaagcctgggaaagcaccaaagctgctgatctatgccgcttctagtctgcagtccggagtgccctctcgattctctggcagtgggtcaggaaccgactttactctgaccatttcaagcctgcagcctgaggatttcgctacatactattgcctgcaggcaaactctttcccccctacctttggcgggggaacaaaagtggagatcaagcgtacggtggcagccccatccgtcttcatttttccaccctctgacgaacagctgaaaagtggcacagccagcgtggtctgtctgctgaacaatttttacccccgcgaagccaaagtgcagtggaaggtcgataacgctctgcagagcgggaattcccaggagtctgtgactgaacaggacagtaaagattcaacctatagcctgtcctctacactgactctgagcaaggcagattacgagaagcacaaagtgtatgcctgcgaagtcacacatcagggactgagttcacctgtgactaagagcttcaatagaggcgagtgttgataa >Ab#1-HC-aa(SEQ ID NO: 403)QLQLQESGPGLVKPSETLSLTCTVSGGSISSSVYYWSWIRQPPGKGLEWIGVIYPSGNTYYSPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARTIYDLFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSKYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSKSCDKTGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSDYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSGECGGGGSGGGGSDIVMTQSPLSLPVTPGEPASISCRSTKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLKISRVEAEDEGVYYCAQNLEIPRTFGGGTKLEIKRTGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGESVKISCKASGYTFTNYGMNWVRQAPGQGLKWMGWINTYTGESTYADDFKGRFAFSLDTSASTAYLQLSSLRSEDTAVYFCARFAIKGDYWGQGTLVTVSS** >Ab#2-HC-aa (SEQ ID NO: 404)QLQLQESGPGLVKPSETLSLTCTVSGGSISSSVYYWSWIRQPPGKGLEWIGVIYPSGNTYYSPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARTIYDLFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSKYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSKSCDKTGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSDYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSGECGGGGSGGGGSQVQLVQSGSELKKPGESVKVSCKASGYTFTNYGMHWVRQAPGQGLKWMGWINTYTGESTYADDFKGRFAFSLDTSASTAYLQISSLKAEDTAVYFCARFAIKGDYWGQGTLVTVSSASTGGGGSGGGGSGGGGSGGGGSDIVMTQSPLSLPVTPGEPASISCRSTKSLLHSDGITYLYWYLQKPGQSPQLLIYQLSNLASGVPDRFSSSGSGTDFTLKISRVEAEDEGVYYCAQNLEIPYTFGQGTKVEIK** >Ab#3-HC-aa (SEQ ID NO: 405)QLQLQESGPGLVKPSETLSLTCTVSGGSISSSVYYWSWIRQPPGKGLEWIGVIYPSGNTYYSPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARTIYDLFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSKYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSKSCDKTGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSDYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSGECGGGGSGGGGSQVQLVQSGSELKKPGESVKVSCKASGYTFTNYGMNWVRQAPGQGLKWMGWINTYTGESTYADDFKGRFAFSLDTSASTAYLQISSLKAEDTAVYFCARFAIKGDYWGQGTLVTVSSASTGGGGSGGGGSGGGGSGGGGSDIVMTQSPLSLPVTPGEPASISCRSTKSLLHSDGITYLYWYLQKPGQSPQLLIYQLSNLASGVPDRFSSSGSGTDFTLKISRVEAEDEGVYYCAQNLEIPRTFGQGTKVEIK** >Ab#4-HC-aa (SEQ ID NO: 406)QLQLQESGPGLVKPSETLSLTCTVSGGSISSSVYYWSWIRQPPGKGLEWIGVIYPSGNTYYSPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARTIYDLFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSKYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSKSCDKTGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSDYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSGECGGGGSGGGGSDIVMTQSPLSLPVTPGEPASISCRSTKSLLHSDGITYLYWYLQKPGQSPQLLIYQLSNLASGVPDRFSSSGSGTDFTLKISRVEAEDEGVYYCAQNLEIPRTFGQGTKVEIKRTGGGGSGGGGSGGGGSGGGGSGQVQLVQSGSELKKPGESVKVSCKASGYTFTNYGMNWVRQAPGQGLKWMGWINTYTGESTYADDFKGRFAFSLDTSASTAYLQISSLKAEDTAVYFCARFAIKGDYWGQGTLVTVSS** >Ab#5-HC-aa (SEQ ID NO: 407)QLQLQESGPGLVKPSETLSLTCTVSGGSISSSVYYWSWIRQPPGKGLEWIGVIYPSGNTYYSPSLKSRVTISVDTSK                                CDR1                     CDR2NQFSLKLSSVTAADTAVYYCARTIYDLFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP                          CDR3VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSKYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSKSCDKTGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSDYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSGECGGGGSGGGGSDIVMTQSPLSLPVTPGEPASISCRSTKSLLHSNGITYLYWYLQKPGQ                                                            CDR4SPQLLIYQMSNLASGVPDRFSSSGSGTDFTLKISRVEAEDEGVYYCAQNLEIPRTFGGGTKLEIKRTGGGGSGGGGS          CDR5                                   CDR6GGGGSGGGGSQVQLVQSGAEVKKPGESVKISCKASGYTFTNYGMNWVRQAPGQGLKWMGWINTYTGESTYADDFKGR                                       CDR7                      CDR8FAFSLDTSASTAYLQLSSLRSEDTAVYFCARFAIKGDYWGQGTLVTVSS**                                  CDR9 >Ab#5-HC-nt (SEQ ID NO: 408)cagctgcagctgcaggagagcggacccggactggtcaagccctccgagacactgtctctgacctgcacagtgtcaggcgggagcatcagctcctctgtctactattggtcttggattcggcagccccctggaaagggcctggaatggatcggcgtgatctacccaagtgggaacacttactattctcccagtctgaaatccagagtgaccatcagtgtcgacacatcaaagaatcagttcagcctgaaactgagttcagtgacagccgctgatactgcagtctactattgtgccaggaccatctatgacctgtttgatatttgggggcagggaactatggtgaccgtcagctccgctagcactaagggaccttccgtgttcccactggccccctcaagcaaatctaccagtgggggaacagcagccctgggctgtctggtgaaggattactttccagagcccgtgaccgtcagttggaactcaggggctctgactagcggagtgcacaccttccccgcagtcctgcagtcctctgggctgtactccctgagttcagtggtcactgtgcccagctcctctctgggaacacagacttatatctgcaacgtgaatcacaagccttccaataccaaagtcgacaagaaagtcgagcctaagtcatgtgataaaacccatacatgcccaagctgtcctgcaccagaatttctgggaggaccaagcgtgttcctgtttccacctaagcctaaagacaccctgatgattagtcgcactccagaggtgacctgtgtggtcgtggacgtcagccaggaggatcccgaagtgcagtttaactggtacgtcgatggcgtggaagtccacaatgctaagacaaaaccaagagaggaacagttcaacagcaagtacagggtcgtgtccgtgctgactgtcctgcatcaggactggctgaacggaaaagagtataagtgcaaagtgagcaataagggcctgcccagttcaatcgagaaaacaatttccaaggcaaaaggccagcctcgggaaccacaggtgtataccctgccacccagcagagaggaaatgacaaagaaccaggtgtcactgagctgtgccgtcaaaggcttttaccctagcgatatcgctgtggagtgggaatccaatgggcagccagaaaacaattataagaccacacctccagtgctggacagcgatggatccttctttctggtgtccaagctgaccgtggacaaatctcggtggcagcagggcaacgtgttctcctgctctgtcatgcatgaggccctgcacaatcattacacccagaaaagtctgtcactgagcaagtcctgtgataaaacaggaggcgggggatctggcgggggaggcagtgggggaggcgggtccggaggaggaggaagcggaggaggaggatccggaggaggcgggtctggaggcgggggaagtggaggaggaggatcttgcccaagttgtccagcacctgagttcctgggaggaccatccgtgttcctgtttccccctaagcccaaagacaccctgatgatcagccggacacctgaagtgacttgcgtcgtggtggacgtgtcccaggaggatccagaggtccagtttaactggtatgtcgatggcgtggaggtccacaatgctaagaccaaacctcgcgaggaacagttcaactctgactaccgagtggtcagtgtgctgacagtcctgcatcaggattggctgaacgggaaggaatacaaatgtaaagtgagcaataagggactgccaagctccatcgagaagaccatcagcaaagccaaaggccagcccagggaacctcaggtgtacactctgccaccctcccgcgaggaaatgaccaagaaccaggtgtctctgtggtgtctggtcaaaggattttatccctctgacatcgccgtggagtgggaaagtaatggccagcctgaaaacaattacaagactacccctccagtgctggactcagatgggagcttctttctgtatagtaagctgaccgtggataaatcacggtggcagcagggaaatgtgttctcttgcagtgtcatgcacgaggccctgcataaccattacacacagaagtcattaagcttatcgggggaatgtggaggcggagggagcggcggaggcggcagcgatatcgtgatgacacagtcacccctgtcgctccctgtgactcccggagagcctgcgtccatctcgtgccggtcaacaaagtccctgctgcacagcaatggaattacgtatttgtactggtatttgcaaaaacccggacagagcccgcaattgttgatctaccagatgtcaaaccttgcgagcggggtcccggatcgctttagctcgtcggggtcaggaactgacttcacactcaaaatctcaagggtcgaggccgaggatgaaggtgtctattactgcgctcagaatctcgaaatcccgcggacctttggtggaggtacgaagctggagatcaagcgaacgggaggcggtgggtccggtggcggtggttccggcggaggtggttcgggaggaggaggtagccaggtgcagcttgtccagtcgggagcagaggtaaagaagccaggagagtcggtgaaaatctcgtgtaaagcctcggggtacacattcacgaactacggcatgaattgggtgagacaagctcccgggcagggcttgaaatggatgggatggattaacacctacacaggggaatccacatatgcggacgacttcaaggggaggttcgcgttttcacttgatacttcagcgagcacggcgtatctccaactctcgtcgcttcgctccgaagataccgcagtatacttttgcgccagattcgcgattaaaggggactattggggacaggggacccttgtcacggtgtcatcatgataa >Ab#6-HC-aa (SEQ ID NO: 409)QLQLQESGPGLVKPSETLSLTCTVSGGSISSSVYYWSWIRQPPGKGLEWIGVIYPSGNTYYSPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARTIYDLFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSKYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSKSCDKTGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSDYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSGECGGGGSGGGGSDIVMTQSPLSLPVTPGEPASISCRSTKSLLHSDGITYLYWYLQKPGQSPQLLIYQLSNLASGVPDRFSSSGSGTDFTLKISRVEAEDEGVYYCAQNLEIPRTFGGGTKLEIKRTGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGESVKISCKASGYTFTNYGMNWVRQAPGQGLKWMGWINTYTGESTYADDFKGRFAFSLDTSASTAYLQLSSLRSEDTAVYFCARFAIKGDYWGQGTLVTVSS** >Ab#7-HC-aa (SEQ ID NO: 410)QLQLQESGPGLVKPSETLSLTCTVSGGSISSSVYYWSWIRQPPGKGLEWIGVIYPSGNTYYSPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARTIYDLFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSKYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSKSCDKTGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSDYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSGECGGGGSGGGGSDIVMTQSPLSLPVTPGEPASISCRSTKSLLHSDGITYLYWYLQKPGQSPQLLIYQLSNLASGVPDRFSSSGSGTDFTLKISRVEAEDEGVYYCAQNLEIPRTFGCGTKLEIKRTGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGESVKISCKASGYTFTNYGMNWVRQAPGQCLKWMGWINTYTGESTYADDFKGRFAFSLDTSASTAYLQLSSLRSEDTAVYFCARFAIKGDYWGQGTLVTVSS** >Ab#7-HC-nt(SEQ ID NO: 411)cagctgcagctgcaggagagcggacccggactggtcaagccctccgagacactgtctctgacctgcacagtgtcaggcgggagcatcagctcctctgtctactattggtcttggattcggcagccccctggaaagggcctggaatggatcggcgtgatctacccaagtgggaacacttactattctcccagtctgaaatccagagtgaccatcagtgtcgacacatcaaagaatcagttcagcctgaaactgagttcagtgacagccgctgatactgcagtctactattgtgccaggaccatctatgacctgtttgatatttgggggcagggaactatggtgaccgtcagctccgctagcactaagggaccttccgtgttcccactggccccctcaagcaaatctaccagtgggggaacagcagccctgggctgtctggtgaaggattactttccagagcccgtgaccgtcagttggaactcaggggctctgactagcggagtgcacaccttccccgcagtcctgcagtcctctgggctgtactccctgagttcagtggtcactgtgcccagctcctctctgggaacacagacttatatctgcaacgtgaatcacaagccttccaataccaaagtcgacaagaaagtcgagcctaagtcatgtgataaaacccatacatgcccaagctgtcctgcaccagaatttctgggaggaccaagcgtgttcctgtttccacctaagcctaaagacaccctgatgattagtcgcactccagaggtgacctgtgtggtcgtggacgtcagccaggaggatcccgaagtgcagtttaactggtacgtcgatggcgtggaagtccacaatgctaagacaaaaccaagagaggaacagttcaacagcaagtacagggtcgtgtccgtgctgactgtcctgcatcaggactggctgaacggaaaagagtataagtgcaaagtgagcaataagggcctgcccagttcaatcgagaaaacaatttccaaggcaaaaggccagcctcgggaaccacaggtgtataccctgccacccagcagagaggaaatgacaaagaaccaggtgtcactgagctgtgccgtcaaaggcttttaccctagcgatatcgctgtggagtgggaatccaatgggcagccagaaaacaattataagaccacacctccagtgctggacagcgatggatccttctttctggtgtccaagctgaccgtggacaaatctcggtggcagcagggcaacgtgttctcctgctctgtcatgcatgaggccctgcacaatcattacacccagaaaagtctgtcactgagcaagtcctgtgataaaacaggaggcgggggatctggcgggggaggcagtgggggaggcgggtccggaggaggaggaagcggaggaggaggatccggaggaggcgggtctggaggcgggggaagtggaggaggaggatcttgcccaagttgtccagcacctgagttcctgggaggaccatccgtgttcctgtttccccctaagcccaaagacaccctgatgatcagccggacacctgaagtgacttgcgtcgtggtggacgtgtcccaggaggatccagaggtccagtttaactggtatgtcgatggcgtggaggtccacaatgctaagaccaaacctcgcgaggaacagttcaactctgactaccgagtggtcagtgtgctgacagtcctgcatcaggattggctgaacgggaaggaatacaaatgtaaagtgagcaataagggactgccaagctccatcgagaagaccatcagcaaagccaaaggccagcccagggaacctcaggtgtacactctgccaccctcccgcgaggaaatgaccaagaaccaggtgtctctgtggtgtctggtcaaaggattttatccctctgacatcgccgtggagtgggaaagtaatggccagcctgaaaacaattacaagactacccctccagtgctggactcagatgggagcttctttctgtatagtaagctgaccgtggataaatcacggtggcagcagggaaatgtgttctcttgcagtgtcatgcacgaggccctgcataaccattacacacagaagtcattaagcttatcgggggaatgtggaggcggagggagcggcggaggcggcagcgatatcgtgatgacacagtcacccctgtcgctccctgtgactcccggagagcctgcgtccatctcgtgccggtcaacaaagtccctgctgcacagcgacggaattacgtatttgtactggtatttgcaaaaacccggacagagcccgcaattgttgatctaccagctctcaaaccttgcgagcggggtcccggatcgctttagctcgtcggggtcaggaactgacttcacactcaaaatctcaagggtcgaggccgaggatgaaggtgtctattactgcgctcagaatctcgaaatcccgcggacctttggttgcggtacgaagctggagatcaagcgaacgggaggcggtgggtccggtggcggtggttccggcggaggtggttcgggaggaggaggtagccaggtgcagcttgtccagtcgggagcagaggtaaagaagccaggagagtcggtgaaaatctcgtgtaaagcctcggggtacacattcacgaactacggcatgaattgggtgagacaagctcccgggcagtgtttgaaatggatgggatggattaacacctacacaggggaatccacatatgcggacgacttcaaggggaggttcgcgttttcacttgatacttcagcgagcacggcgtatctccaactgtcgtcgcttcgctccgaagataccgcagtatacttttgcgccagattcgcgattaaaggggactattggggacaggggacccttgtcacggtgtcatcatgataa >Ab#8-HC-aa (SEQ ID NO: 412)QLQLQESGPGLVKPSETLSLTCTVSGGSISSSVYYWSWIRQPPGKGLEWIGVIYPSGNTYYSPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARTIYDLFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSKYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSKSCDKTGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSDYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSGECGGGGSGGGGSQVQLVQSGAEVKKPGESVKISCKASGYTFTNYGMNWVRQAPGQGLKWMGWINTYTGESTYADDFKGRFAFSLDTSASTAYLQLSSLRSEDTAVYFCARFAIKGDYWGQGTLVTVSSASTGGGGSGGGGSGGGGSGGGGSDIVMTQSPLSLPVTPGEPASISCRSTKSLLHSDGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLKISRVEAEDEGVYYCAQNLEIPRTFGGGTKLEIKRT** >Ab#9-HC-aa (SEQ ID NO: 413)QLQLQESGPGLVKPSETLSLTCTVSGGSISSSVYYWSWIRQPPGKGLEWIGVIYPSGNTYYSPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARTIYDLFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSKYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSKSCDKTGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSDYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSGECGGGGSGGGGSQVQLQQSGGGLVKPGESVKISCAASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGESTYADDFKGRFTESLETSASAAYLQINSLRPEDTAVYFCARFAIKGDYWGQGTTVTVSSASTGGGGSGGGGSGGGGSGGGGSGDIVMTQSPSSLSASVGDKATITCRSTKSLLHSNGITYLYWYLQKPGKAPKLLIYQMSNLASGVPDRFSSSGSGTEFTLTISSVQPEDEGTYYCAQNLEIPRTFGQGTKLEIK** >Ab#10-HC-aa(SEQ ID NO: 414)QLQLQESGPGLVKPSETLSLTCTVSGGSISSSVYYWSWIRQPPGKGLEWIGVIYPSGNTYYSPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARTIYDLFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSKYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSKSCDKTGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSDYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSGECGGGGSGGGGSQVQLQQSGGGLVKPGESVKISCAASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGESTYADDFKGRFTESLETSASAAYLQINSLRPEDTAVYFCARFAIKGDYWGQGTTVTVSSASTGGGGSGGGGSGGGGSGGGGSGDIVMTQSPSSLSASVGDKATITCRSTKSLLHSDGITYLYWYLQKPGKAPKLLIYQLSNLASGVPDRFSSSGSGTEFTLTISSVQPEDEGTYYCAQNLEIPRTFGQGTKLEIK** >Ab#11-HC-aa (SEQ ID NO: 415)QLQLQESGPGLVKPSETLSLTCTVSGGSISSSVYYWSWIRQPPGKGLEWIGVIYPSGNTYYSPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARTIYDLFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSKYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSKSCDKTGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSDYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSGECGGGGSGGGGSDIVMTQSPSSLSASVGDKATITCRSTKSLLHSNGITYLYWYLQKPGKAPKLLIYQMSNLASGVPDRFSSSGSGTEFTLTISSVQPEDEGTYYCAQNLEIPRTFGQGTKLEIKRTGGGGSGGGGSGGGGSGGGGSGQVQLQQSGGGLVKPGESVKISCAASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGESTYADDFKGRFTESLETSASAAYLQINSLRPEDTAVYFCARFAIKGDYWGQGTTVTVSS** >Ab#12-HC-aa (SEQ ID NO: 416)QLQLQESGPGLVKPSETLSLTCTVSGGSISSSVYYWSWIRQPPGKGLEWIGVIYPSGNTYYSPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARTIYDLFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSKYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSKSCDKTGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSDYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSGECGGGGSGGGGSDIVMTQSPSSLSASVGDKATITCRSTKSLLHSDGITYLYWYLQKPGKAPKLLIYQLSNLASGVPDRFSSSGSGTEFTLTISSVQPEDEGTYYCAQNLEIPRTFGQGTKLEIKRTGGGGSGGGGSGGGGSGGGGSGQVQLQQSGGGLVKPGESVKISCAASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGESTYADDFKGRFTESLETSASAAYLQINSLRPEDTAVYFCARFAIKGDYWGQGTTVTVSS** >Ab#13-HC-aa (SEQ ID NO: 417)QLQLQESGPGLVKPSETLSLTCTVSGGSISSSVYYWSWIRQPPGKGLEWIGVIYPSGNTYYSPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARTIYDLFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSKYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSKSCDKTGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSDYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSGECGGGGSGGGGSDIVMTQSPSSLSASVGDKATITCRSTKSLLHSNGITYLYWYLQKPGKAPKLLIYQMSNLASGVPDRFSSSGSGTEFTLTISSVQPEDEGTYYCAQNLEIPRTFGQGTKLEIKRTPSHNSHQVPSAGGPTANSGTSGSQVQLQQSGGGLVKPGESVKISCAASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGESTYADDFKGRFTESLETSASAAYLQINSLRPEDTAVYFCARFAIKGDYWGQGTTVTVSS** >Ab#3-HC-nt (SEQ ID NO: 418)cagctgcagctgcaggagagcggacccggactggtcaagccctccgagacactgtctctgacctgcacagtgtcaggcgggagcatcagctcctctgtctactattggtcttggattcggcagccccctggaaagggcctggaatggatcggcgtgatctacccaagtgggaacacttactattctcccagtctgaaatccagagtgaccatcagtgtcgacacatcaaagaatcagttcagcctgaaactgagttcagtgacagccgctgatactgcagtctactattgtgccaggaccatctatgacctgtttgatatttgggggcagggaactatggtgaccgtcagctccgctagcactaagggaccttccgtgttcccactggccccctcaagcaaatctaccagtgggggaacagcagccctgggctgtctggtgaaggattactttccagagcccgtgaccgtcagttggaactcaggggctctgactagcggagtgcacaccttccccgcagtcctgcagtcctctgggctgtactccctgagttcagtggtcactgtgcccagctcctctctgggaacacagacttatatctgcaacgtgaatcacaagccttccaataccaaagtcgacaagaaagtcgagcctaagtcatgtgataaaacccatacatgcccaagctgtcctgcaccagaatttctgggaggaccaagcgtgttcctgtttccacctaagcctaaagacaccctgatgattagtcgcactccagaggtgacctgtgtggtcgtggacgtcagccaggaggatcccgaagtgcagtttaactggtacgtcgatggcgtggaagtccacaatgctaagacaaaaccaagagaggaacagttcaacagcaagtacagggtcgtgtccgtgctgactgtcctgcatcaggactggctgaacggaaaagagtataagtgcaaagtgagcaataagggcctgcccagttcaatcgagaaaacaatttccaaggcaaaaggccagcctcgggaaccacaggtgtataccctgccacccagcagagaggaaatgacaaagaaccaggtgtcactgagctgtgccgtcaaaggcttttaccctagcgatatcgctgtggagtgggaatccaatgggcagccagaaaacaattataagaccacacctccagtgctggacagcgatggatccttctttctggtgtccaagctgaccgtggacaaatctcggtggcagcagggcaacgtgttctcctgctctgtcatgcatgaggccctgcacaatcattacacccagaaaagtctgtcactgagcaagtcctgtgataaaacaggaggcgggggatctggcgggggaggcagtgggggaggcgggtccggaggaggaggaagcggaggaggaggatccggaggaggcgggtctggaggcgggggaagtggaggaggaggatcttgcccaagttgtccagcacctgagttcctgggaggaccatccgtgttcctgtttccccctaagcccaaagacaccctgatgatcagccggacacctgaagtgacttgcgtcgtggtggacgtgtcccaggaggatccagaggtccagtttaactggtatgtcgatggcgtggaggtccacaatgctaagaccaaacctcgcgaggaacagttcaactctgactaccgagtggtcagtgtgctgacagtcctgcatcaggattggctgaacgggaaggaatacaaatgtaaagtgagcaataagggactgccaagctccatcgagaagaccatcagcaaagccaaaggccagcccagggaacctcaggtgtacactctgccaccctcccgcgaggaaatgaccaagaaccaggtgtctctgtggtgtctggtcaaaggattttatccctctgacatcgccgtggagtgggaaagtaatggccagcctgaaaacaattacaagactacccctccagtgctggactcagatgggagcttctttctgtatagtaagctgaccgtggataaatcacggtggcagcagggaaatgtgttctcttgcagtgtcatgcacgaggccctgcataaccattacacacagaagtcattaagcttatcgggagagtgcggtggcggagggagcggaggaggcgggtcggacatcgtcatgacccagtcaccgtcctcactgtcggcgtcggtgggtgataaggccacaattacatgccgcagcacgaaatcactgctccactccaatgggattacatatctctattggtatctccaaaaacccggaaaggcacctaagttgctgatctaccagatgtcgaacttggcatcgggagtacccgataggttctcgtcgtcgggaagcggcacggagttcacgctcaccatttcctcagtccagccggaggacgaaggaacttactactgcgctcagaatcttgaaatcccgcgcacatttggacaagggacgaaacttgaaatcaagcgaactccgtcccacaacagccatcaagtgccctcggcgggagggcccaccgccaattcggggacatcagggagccaggtacagttgcagcagtcgggaggcgggctggtaaaacctggtgaaagcgtcaagatctcatgtgcagcctcagggtatacgttcaccaattacgggatgaactgggtgaagcaggcgccagggaaaggtcttaagtggatgggatggatcaacacttacacgggagagtccacatacgcggatgactttaaggggcggttcacgttttcgttggagacttcagcgtccgctgcctacctccaaatcaactcccttagacccgaggacacagcggtctatttctgtgcgcggtttgccattaaaggtgattattggggacagggtacgactgtgaccgtgtccagctgataacMet-his (bold is signal sequence) (SEQ ID NO: 419)MGAPAVLAPGILVLLFTLVQRSNGECKEALAKSEMNVNMKYQLPNFTAETPIQNVILHEHHIFLGATNYIYVLNEEDLQKVAEYKTGPVLEHPDCFPCQDCSSKANLSGGVWKDNINMALVVDTYYDDQLISCGSVNRGTCQRHVFPHNHTADIQSEVHCIFSPQIEEPSQCPDCVVSALGAKVLSSVKDRFINFFVGNTINSSYFPDHPLHSISVRRLKETKDGFMFLTDQSYIDVLPEFRDSYPIKYVHAFESNNFIYFLTVQRETLDAQTFHTRIIRFCSINSGLHSYMEMPLECILTEKRKKRSTKKEVFNILQAAYVSKPGAQLARQIGASLNDDILFGVFAQSKPDSAEPMDRSAMCAFPIKYVNDFFNKIVNKNNVRCLQHFYGPNHEHCFNRTLLRNSSGCEARRDEYRTEFTTALQRVDLFMGQFSEVLLTSISTFIKGDLTIANLGTSEGRFMQVVVSRSGPSTPHVNFLLDSHPVSPEVIVEHTLNQNGYTLVITGKKITKIPLNGLGCRHFQSCSQCLSAPPFVQCGWCHDKCVRSEECLSGTWTQQICLPAIYKVFPNSAPLEGGTRLTICGWDFGFRRNNKFDLKKTRVLLGNESCTLTLSESTMNTLKCTVGPAMNKHFNMSIIISNGHGTTQYSTFSYVDPVITSISPKYGPMAGGTLLTLTGNYLNSGNSRHISIGGKTCTLKSVSNSILECYTPAQTISTEFAVKLKIDLANRETSIFSYREDPIVYEIHPTKSFISGGSTITGVGKNLNSVSVPRMVINVHEAGRNFTVACQHRSNSEIICCTTPSLQQLNLQLPLKTKAFFMLDGILSKYFDLIYVHNPVFKPFEKPVMISMGNENVLEIKGNDIDPEAVKGEVLKVGNKSCENIHLHSEAVLCTVPNDLLKLNSELNIEWKQAISSTVLGKVIVQPDQNFTGGGGSHHHHHH**Human EpCAM-Fc-his tagged (bold is signal sequence)  (SEQ ID NO: 420)MGWSLILLFLVAVATRVLSQEECVCENYKLAVNCFVNNNRQCQCTSVGAQNTVICSKLAAKCLVMKAEMNGSKLGRRAKPEGALQNNDGLYDPDCDESGLFKAKQCNGTSMCWCVNTAGVRRTDKDTEITCSERVRTYWIIIELKHKAREKPYDSKSLRTALQKEITTRYQLDPKFITSILYENNVITIDLVQNSSQKTQNDVDIADVAYYFEKDVKGESLFHSKKMDLTVNGEQLDLDPGQTLIYYVDEKAPEFSMQGLKIEGRMDPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSHHHHHH** Anti-c-Met-OA-5D5-HC-aa(SEQ ID NO: 421)EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTRFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSSAnti-c-Met-OA-5D5-LC-aa (SEQ ID NO: 422)DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIKRRESERVED: SEQ ID NO:423, SEQ ID NO: 424 >Ab#X1-OA-5D5  (SEQ ID NO: 425)EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTRFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSKYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSKSCDKTGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSDYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSGECGGGGSGGGGSDIVMTQSPLSLPVTPGEPASISCRSTKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLKISRVEAEDEGVYYCAQNLEIPRTFGGGTKLEIKRTGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGESVKISCKASGYTFTNYGMNWVRQAPGQGLKWMGWINTYTGESTYADDFKGRFAFSLDTSASTAYLQLSSLRSEDTAVYFCARFAIKGDYWGQGTLVTVSS** >Ab#X2-OA-5D5 (SEQ ID NO: 426)EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTRFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSKYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSKSCDKTGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSDYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSGECGGGGSGGGGSQVQLVQSGSELKKPGESVKVSCKASGYTFTNYGMHWVRQAPGQGLKWMGWINTYTGESTYADDFKGRFAFSLDTSASTAYLQISSLKAEDTAVYFCARFAIKGDYWGQGTLVTVSSASTGGGGSGGGGSGGGGSGGGGSDIVMTQSPLSLPVTPGEPASISCRSTKSLLHSDGITYLYWYLQKPGQSPQLLIYQLSNLASGVPDRFSSSGSGTDFTLKISRVEAEDEGVYYCAQNLEIPYTFGQGTKVEIK** >Ab#X3-OA-5D5(SEQ ID NO: 427)EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTRFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSKYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSKSCDKTGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSDYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSGECGGGGSGGGGSQVQLVQSGSELKKPGESVKVSCKASGYTFTNYGMNWVRQAPGQGLKWMGWINTYTGESTYADDFKGRFAFSLDTSASTAYLQISSLKAEDTAVYFCARFAIKGDYWGQGTLVTVSSASTGGGGSGGGGSGGGGSGGGGSDIVMTQSPLSLPVTPGEPASISCRSTKSLLHSDGITYLYWYLQKPGQSPQLLIYQLSNLASGVPDRFSSSGSGTDFTLKISRVEAEDEGVYYCAQNLEIPRTFGQGTKVEIK** >Ab#X4-OA-5D5 (SEQ ID NO: 428)EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTRFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSKYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSKSCDKTGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSDYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSGECGGGGSGGGGSDIVMTQSPLSLPVTPGEPASISCRSTKSLLHSDGITYLYWYLQKPGQSPQLLIYQLSNLASGVPDRFSSSGSGTDFTLKISRVEAEDEGVYYCAQNLEIPRTFGQGTKVEIKRTGGGGSGGGGSGGGGSGGGGSGQVQLVQSGSELKKPGESVKVSCKASGYTFTNYGMNWVRQAPGQGLKWMGWINTYTGESTYADDFKGRFAFSLDTSASTAYLQISSLKAEDTAVYFCARFAIKGDYWGQGTLVTVSS** >Ab#X5-OA-5D5 (SEQ ID NO: 429)EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTRFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSKYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSKSCDKTGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSDYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSGECGGGGSGGGGSDIVMTQSPLSLPVTPGEPASISCRSTKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLKISRVEAEDEGVYYCAQNLEIPRTFGGGTKLEIKRTGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGESVKISCKASGYTFTNYGMNWVRQAPGQGLKWMGWINTYTGESTYADDFKGRFAFSLDTSASTAYLQLSSLRSEDTAVYFCARFAIKGDYWGQGTLVTVSS** >Ab#X6-OA-5D5 (SEQ ID NO: 430)EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTRFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPSCPAPEFLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSKYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSKSCDKTGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSDYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSGECGGGGSGGGGSDIVMTQSPLSLPVTPGEPASISCRSTKSLLHSDGITYLYWYLQKPGQSPQLLIYQLSNLASGVPDRFSSSGSGTDFTLKISRVEAEDEGVYYCAQNLEIPRTFGGGTKLEIKRTGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGESVKISCKASGYTFTNYGMNWVRQAPGQGLKWMGWINTYTGESTYADDFKGRFAFSLDTSASTAYLQLSSLRSEDTAVYFCARFAIKGDYWGQGTLVTVSS** >Ab#X7-OA-5D5(SEQ ID NO: 431)EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTRFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSKYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSKSCDKTGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSDYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSGECGGGGSGGGGSDIVMTQSPLSLPVTPGEPASISCRSTKSLLHSDGITYLYWYLQKPGQSPQLLIYQLSNLASGVPDRFSSSGSGTDFTLKISRVEAEDEGVYYCAQNLEIPRTFGCGTKLEIKRTGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGESVKISCKASGYTFTNYGMNWVRQAPGQCLKWMGWINTYTGESTYADDFKGRFAFSLDTSASTAYLQLSSLRSEDTAVYFCARFAIKGDYWGQGTLVTVSS** >Ab#X8-OA-5D5 (SEQ ID NO: 432)EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTRFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSKYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSKSCDKTGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSDYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSGECGGGGSGGGGSQVQLVQSGAEVKKPGESVKISCKASGYTFTNYGMNWVRQAPGQGLKWMGWINTYTGESTYADDFKGRFAFSLDTSASTAYLQLSSLRSEDTAVYFCARFAIKGDYWGQGTLVTVSSASTGGGGSGGGGSGGGGSGGGGSDIVMTQSPLSLPVTPGEPASISCRSTKSLLHSDGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLKISRVEAEDEGVYYCAQNLEIPRTFGGGTKLEIKRT** >Ab#X9-OA-5D5 (SEQ ID NO: 433)EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTRFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSKYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSKSCDKTGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSDYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSGECGGGGSGGGGSQVQLQQSGGGLVKPGESVKISCAASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGESTYADDFKGRFTFSLETSASAAYLQINSLRPEDTAVYFCARFAIKGDYWGQGTTVTVSSASTGGGGSGGGGSGGGGSGGGGSGDIVMTQSPSSLSASVGDKATITCRSTKSLLHSNGITYLYWYLQKPGKAPKLLIYQMSNLASGVPDRFSSSGSGTEFTLTISSVQPEDEGTYYCAQNLEIPRTFGQGTKLEIK** >Ab#X10-OA-5D5(SEQ ID NO: 434)EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTRFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSKYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSKSCDKTGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSDYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSGECGGGGSGGGGSQVQLQQSGGGLVKPGESVKISCAASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGESTYADDFKGRFTFSLETSASAAYLQINSLRPEDTAVYFCARFAIKGDYWGQGTTVTVSSASTGGGGSGGGGSGGGGSGGGGSGDIVMTQSPSSLSASVGDKATITCRSTKSLLHSDGITYLYWYLQKPGKAPKLLIYQLSNLASGVPDRFSSSGSGTEFTLTISSVQPEDEGTYYCAQNLEIPRTFGQGTKLEIK** >Ab#X11-OA-5D5 (SEQ ID NO: 435)EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTRFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSKYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSKSCDKTGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSDYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSGECGGGGSGGGGSDIVMTQSPSSLSASVGDKATITCRSTKSLLHSNGITYLYWYLQKPGKAPKLLIYQMSNLASGVPDRFSSSGSGTEFTLTISSVQPEDEGTYYCAQNLEIPRTFGQGTKLEIKRTGGGGSGGGGSGGGGSGGGGSGQVQLQQSGGGLVKPGESVKISCAASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGESTYADDFKGRFTESLETSASAAYLQINSLRPEDTAVYFCARFAIKGDYWGQGTTVTVSS** >Ab#X12-OA-5D5 (SEQ ID NO: 436)EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTRFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSKYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSKSCDKTGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSDYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSGECGGGGSGGGGSDIVMTQSPSSLSASVGDKATITCRSTKSLLHSDGITYLYWYLQKPGKAPKLLIYQLSNLASGVPDRFSSSGSGTEFTLTISSVQPEDEGTYYCAQNLEIPRTFGQGTKLEIKRTGGGGSGGGGSGGGGSGGGGSGQVQLQQSGGGLVKPGESVKISCAASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGESTYADDFKGRFTESLETSASAAYLQINSLRPEDTAVYFCARFAIKGDYWGQGTTVTVSS** >Ab#X13-OA-5D5 (SEQ ID NO: 437)EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTRFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSKYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSKSCDKTGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSDYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSGECGGGGSGGGGSDIVMTQSPSSLSASVGDKATITCRSTKSLLHSNGITYLYWYLQKPGKAPKLLIYQMSNLASGVPDRFSSSGSGTEFTLTISSVQPEDEGTYYCAQNLEIPRTFGQGTKLEIKRTPSHNSHQVPSAGGPTANSGTSGSQVQLQQSGGGLVKPGESVKISCAASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGESTYADDFKGRFTESLETSASAAYLQINSLRPEDTAVYFCARFAIKGDYWGQGTTVTVSS** >Anti-EpCAM-HC1 aa  (SEQ ID NO: 438)QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLKWMGWINTYTGESTYADDFKGRVTITADTSASTAYMELSSLRSEDTAVYYCARFAIKGDYWGQGTLVTVSS >Anti-EpCAM-HC1 nt (SEQ ID NO: 439)caagtgcagttggtccagagcggtgcggaggtaaagaaacccggtgcatccgtgaaggtgtcgtgcaaagcctccgggtatacgttcacgaactatgggatgaactgggtcagacaagcaccgggtcagggactcaaatggatggggtggatcaatacatacacaggggaatcgacctacgcggatgactttaagggaagggtcaccattacggcggacacctcggcatcgactgcgtatatggaactctcatcacttcgctcggaggacacagccgtctactattgtgcgcggtttgcgatcaagggagattactggggacagggaactttggtaacagtatcaagc >Anti-EpCAM-HC2 aa(SEQ ID NO: 440)QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLKWMGWINTYTGESTYADDFKGRVTITLDTSASTAYMELSSLRSEDTAVYFCARFAIKGDYWGQGTLVTVSS >Anti-EpCAM-HC2 nt(SEQ ID NO: 441)caagtgcagttggtccagagcggtgcggaggtaaagaaacccggtgcatccgtgaaggtgtcgtgcaaagcctccgggtatacgttcacgaactatgggatgaactgggtcagacaagcaccgggtcagggactcaaatggatggggtggatcaatacatacacaggggaatcgacctacgcggatgactttaagggaagggtcaccattacgttggacacctcggcatcgactgcgtatatggaactctcatcacttcgctcggaggacacagccgtctacttctgtgcgcggtttgcgatcaagggagattactggggacagggaactttggtaacagtatcaagc >anti-EpCAM-HC3 aa(SEQ ID NO: 442)QVQLQQSGGGLVKPGGSVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGESTYADDFKGRFAFSLETSASAAYLQINSLRPEDTAVYFCARFAIKGDYWGQGTLVTVSS >Anti-EpCAM-HC3 nt(SEQ ID NO: 443)caggtacagctgcagcaaagcgggggaggactcgtgaagcctggtggttcggtcaaaatctcgtgtaaagcgtcagggtacaccttcacaaactatggtatgaactgggtgaaacaggcacccggaaagggtcttaagtggatgggctggatcaatacctacacgggggagtcgacatatgcggacgactttaaaggacggttcgcgttttcgttggagactagcgcctccgctgcctacctccaaatcaatagccttaggccggaagatacggcggtctacttttgcgcaagatttgccattaagggggattattggggccaggggacgctggtgacagtcagctccgctagc >anti-EpCAM-HC4 aa(SEQ ID NO: 444)QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLKWMGWINTYTGESTYADDFKGRFAFSLDTSASTAYMELSSLRSEDTAVYFCARFAIKGDYWGQGTLVTVSS >anti-EpCAM-HC4 nt(SEQ ID NO: 445)caggtccagcttgtgcagtccggagccgaagtcaagaagccgggagccagcgtaaaggtgtcatgtaaggcgtcggggtatacattcacgaactacggtatgaattgggtgcgccaagctcccggacagggtttgaaatggatggggtggatcaacacgtatacaggggaatcaacttacgccgacgacttcaagggaaggttcgcattttcgttggatacatcggcgtccacggcgtacatggagctgtcaagcctgcggtcggaggacacggcggtatacttctgcgcaagatttgctatcaaaggtgattattgggggcagggaaccctggtaaccgtgagcagc >anti-EpCAM-HC5 aa(SEQ ID NO: 446)QVQLVQSGSELKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLKWMGWINTYTGESTYADDFKGRFAFSLDTSASTAYLQISSLKAEDTAVYFCARFAIKGDYWGQGTLVTVSS >anti-EpCAM-HC5 nt(SEQ ID NO: 447)caggtccagctggtgcagtcaggttcagagctgaagaagcccggagcgtccgtcaaagtgtcatgcaaggcctcgggttacacgtttacgaactacggtatgaattgggtccgccaggctccgggccagggactgaaatggatgggatggatcaacacatatactggtgaatccacgtatgcggacgactttaaggggagattcgcgttcagccttgatacatcggcgtcgaccgcgtacctccaaatctcgtccttgaaagcagaggacactgcagtatacttttgtgcccggttcgctatcaagggagattattggggccaagggaccttggtgacagtgtccagc >anti-EpCAM-HC6 aa(SEQ ID NO: 448)QVQLVQSGAEVKKPGASVKISCKASGYTFTNYGMNWVRQAPGQGLKWMGWINTYTGESTYADDFKGRFTITLDTSASTAYMELSSLRSEDTAVYYCARFAIKGDYWGQGTLVTVSS >anti-EpCAM-HC6 nt(SEQ ID NO: 449)caagtccagttggtacaatcgggtgccgaggtaaagaaaccgggagcgtcggtcaaaatcagctgcaaggcctcaggctatacctttacaaactacggcatgaattgggtgagacaggcacccgggcagggattgaaatggatgggttggatcaacacgtatacaggggagtccacctatgcagatgactttaaggggcgcttcactatcacgctcgacacgtccgcgtcgacggcgtacatggaactgtcatcgcttcggagcgaagatacagccgtgtactattgtgctaggtttgcgattaagggagactactggggacagggaaccctcgtaactgtgtcatca >anti-EpCAM-HC7 aa(SEQ ID NO: 450)QVQLVQSGAEVKKPGASVKISCKASGYTFTNYGMNWVRQAPGQGLKWMGWINTYTGESTYADDFKGRFAFSLDTSASTAYLQLSSLRSEDTAVYFCARFAIKGDYWGQGTLVTVSS >anti-EpCAM-HC7 nt(SEQ ID NO: 451)caagtgcagctggtacaatcaggtgcggaggtgaaaaagccgggtgcctccgtcaagatttcatgcaaggcctcgggatacacattcaccaactatgggatgaattgggtgaggcaggctcccggtcaggggttgaaatggatggggtggattaacacatatacgggagaaagcacgtatgcggacgacttcaaggggcgcttcgcgttttccctggatacttcggcatcgactgcgtacctccagttgtcgtcgcttagatccgaggacacggccgtctacttctgtgcacggtttgcaatcaagggggattactggggacagggaacgcttgtaaccgtaagctca >Anti-EpCAM-LC1 aa(SEQ ID NO: 452)DIVMTQSPLSLPVTPGEPASISCRSTKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLEIPRTFGQGTKVEIK >Anti-EpCAM-LC1 nt(SEQ ID NO: 453)gacattgtcatgacgcagtcgccgctctcgctcccggtcacaccgggagaacccgcgtccatttcatgcagatcgacaaagtcactcctccattcaaatggaatcacttacttgtactggtatcttcaaaaacccggtcagtcaccacagttgctcatctaccaaatgtccaatttggcttcgggagtgcccgaccgattcagcggttcggggagcggtacggattttacgttgaagatcagcagggtagaggcggaggacgtgggggtgtactattgtgcacagaaccttgaaattccacgcacctttggacaaggcaccaaggtcgaaatcaagcgtacggtggcggcaccttcagtgttcatctttcccccttccgatgaacagctcaaaagcgggactgcatccgtagtctgtcttttgaacaacttctatcccagagaggcgaaagtacagtggaaggtggacaacgcccttcaatcaggcaatagccaggagtcggtgacggagcaggattccaaagatagcacatactcgctttcatccactttgacattgtcgaaagcggactacgaaaagcacaaggtctatgcgtgcgaggtgacgcaccagggactttcgtcgccggtaaccaagtcgttcaatcgcggggagtgctgataa >Anti-EpCAM-LC2 aa(SEQ ID NO: 454)DIVMTQSPSSLSASVGDKATITCRSTKSLLHSNGITYLYWYLQKPGKAPKLLIYQMSNLASGVPDRFSSSGSGTDFTLRISSVQPEDFATYYCAQNLEIPRTFGGGTKLEIK >Anti-EpCAM-LC2 nt(SEQ ID NO: 455)gacattgtcatgacccagtcgccgtcatccctttcagcgagcgtgggggataaggcaacgatcacgtgtcgaagcaccaaatccttgctgcacagcaacgggattacgtatttgtactggtatttgcaaaaacccgggaaagccccgaagctcctcatctaccagatgtcgaatctggcgtcgggggtgccagatcggttctccagcagcgggtcgggtacagacttcacactccggatctcatcagtgcagcccgaggactttgcgacctactattgtgcccaaaatcttgaaatcccgagaacgttcggaggtggcacgaaattggagattaaa >anti-EpCAM-LC3 aa(SEQ ID NO: 456)DIVMTQSPLSLPVTPGEPASISCRSTKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCAQNLEIPRTFGQGTKVEIK >anti-EpCAM-LC3 nt(SEQ ID NO: 457)gatatcgtaatgacccaatcgccgttgtcgcttccagtcacacccggggagcctgcttcgattagctgcagatcaacgaagtcgctcctccattcaaacgggattacgtatttgtactggtatcttcaaaagccgggtcagagcccgcagctgctgatctaccagatgtccaacttggcctcgggcgtccccgaccggtttagcagcagcgggtcgggaacggacttcactctcaagatctcaagggtcgaagcggaagatgtgggtgtgtattactgtgcgcagaatcttgagattccccgaacattcggtcagggaaccaaagtcgagatcaag >anti-EpCAM-LC4 aa (SEQ ID NO: 458)DIVMTQSPLSLPVTPGEPASISCRSTKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCAQNLEIPRTFGGGTKLEIK >anti-EpCAM-LC4 NT (SEQ ID NO: 459)Gacatcgtgatgacgcagtccccactgtcgctccctgtaacaccaggggagcccgcttccatttcgtgtaggtcaacgaagtccttgcttcatagcaatgggatcacttacttgtactggtatctccaaaaaccgggtcagtcccctcagttgctgatctaccagatgtcgaaccttgcgagcggtgtcccggatcgattttcatcgtccggatcgggaaccgacttcacactgaagattagccgcgtggaggccgaagatgtcggggtctactactgtgctcaaaaccttgagattccccggacgtttggaggcggcacgaagctggaaatcaag >Anti-EpCAM scFv-v1 aa(SEQ ID NO: 460)QVQLVQSGSELKKPGESVKVSCKASGYTFTNYGMNWVRQAPGQCLKWMGWINTYTGESTYADDFKGRFAFSLDTSASTAYLQISSLKAEDTAVYFCARFAIKGDYWGQGTLVTVSSASTGGGGSGGGGSGGGGSGGGGSDIVMTQSPLSLPVTPGEPASISCRSTKSLLHSDGITYLYWYLQKPGQSPQLLIYQLSNLASGVPDRFSSSGSGT                               *                    *DFTLKISRVEAEDEGVYYCAQNLEIPRTFGCGTKVEIK** >Anti-EpCAM scFv-v1 nt(SEQ ID NO: 461)caggtacaactcgtgcagtcggggtcggagcttaagaagccgggagagtcggtaaaagtgtcgtgtaaagcctccgggtatacctttacgaattacggcatgaactgggtccggcaggcacccggtcagtgtctcaagtggatgggctggatcaatacgtacacgggcgaatccacttatgcagatgactttaagggaagattcgcgttctcattggacacatcagcgtcaacggcgtatcttcaaatttcgtccttgaaagcggaggacacggcggtctatttctgcgcacgattcgctatcaaaggagattactggggtcaggggacacttgtgacagtgtcgagcgccagcaccggaggtggagggagcggaggaggaggtagcggtggcggtggctcgggtggaggtggatcggacatcgtcatgactcagagccctctgtcactgcccgtaactcccggggagcccgcctccattagctgtcggtcgacaaagtccttgctccactcagatggaatcacgtatttgtactggtacttgcaaaagcctggacagtcgccacagctccttatctaccagttgtccaacctggcgtcgggagtaccggaccgcttttcatcatccgggtcaggcaccgatttcacactgaagattagccgcgtggaggcggaggacgaaggggtctactactgcgcccagaacctcgaaattccgaggacctttggatgcggtactaaagtggaaatcaagtgataa >Anti-EpCAM scFv-v2 aa (SEQ ID NO: 462)QVQLVQSGSELKKPGESVKVSCKASGYTFTNYGMHWVRQAPGQGLKWMGWINTYTGESTYADDFKGRFAFSLDTSASTAYLQISSLKAEDTAVYFCARFAIKGDYWGQGTLVTVSSASTGGGGSGGGGSGGGGSGGGGSDIVMTQSPLSLPVTPGEPASISCRSTKSLLHSDGITYLYWYLQKPGQSPQLLIYQLSNLASGVPDRFSSSGSGT                             *                      *DFTLKISRVEAEDEGVYYCAQNLEIPYTFGQGTKVEIK** >Anti-EpCAM scFv-v2 nt(SEQ ID NO: 463)caggtacaactcgtgcagtcggggtcggagcttaagaagccgggagagtcggtaaaagtgtcgtgtaaagcctccgggtatacctttacgaattacggcatgcattgggtccggcaggcacccggtcaggggctcaagtggatgggctggatcaatacgtacacgggcgaatccacttatgcagatgactttaagggaagattcgcgttctcattggacacatcagcgtcaacggcgtatcttcaaatttcgtccttgaaagcggaggacacggcggtctatttctgcgcacgattcgctatcaaaggagattactggggtcaggggacacttgtgacagtgtcgagcgccagcaccggaggtggagggagcggaggaggaggtagcggtggcggtggctcgggtggaggtggatcggacatcgtcatgactcagagccctctgtcactgcccgtaactcccggggagcccgcctccattagctgtcggtcgacaaagtccttgctccactcagatggaatcacgtatttgtactggtacttgcaaaagcctggacagtcgccacagctccttatctaccagttgtccaacctggcgtcgggagtaccggaccgcttttcatcatccgggtcaggcaccgatttcacactgaagattagccgcgtggaggcggaggacgaaggggtctactactgcgcccagaacctcgaaattccgtacacctttggacaaggtactaaagtggaaatcaagtgataa >Anti-EpCAM scFv-v3 aa  (SEQ ID NO: 464)QVQLVQSGSELKKPGESVKVSCKASGYTFTNYGMNWVRQAPGQGLKWMGWINTYTGESTYADDFKGRFAFSLDTSASTAYLQISSLKAEDTAVYFCARFAIKGDYWGQGTLVTVSSASTGGGGSGGGGSGGGGSGGGGSDIVMTQSPLSLPVTPGEPASISCRSTKSLLHSDGITYLYWYLQKPGQSPQLLIYQLSNLASGVPDRFSSSGSGT                             *                      *DFTLKISRVEAEDEGVYYCAQNLEIPRTFGQGTKVEIK ** >Anti-EpCAM scFv-v3 nt (SEQ ID NO: 465)caggtacaactcgtgcagtcggggtcggagcttaagaagccgggagagtcggtaaaagtgtcgtgtaaagcctccgggtatacctttacgaattacggcatgaactgggtccggcaggcacccggtcaggggctcaagtggatgggctggatcaatacgtacacgggcgaatccacttatgcagatgactttaagggaagattcgcgttctcattggacacatcagcgtcaacggcgtatcttcaaatttcgtccttgaaagcggaggacacggcggtctatttctgcgcacgattcgctatcaaaggagattactggggtcaggggacacttgtgacagtgtcgagcgccagcaccggaggtggagggagcggaggaggaggtagcggtggcggtggctcgggtggaggtggatcggacatcgtcatgactcagagccctctgtcactgcccgtaactcccggggagcccgcctccattagctgtcggtcgacaaagtccttgctccactcagatggaatcacgtatttgtactggtacttgcaaaagcctggacagtcgccacagctccttatctaccagttgtccaacctggcgtcgggagtaccggaccgcttttcatcatccgggtcaggcaccgatttcacactgaagattagccgcgtggaggcggaggacgaaggggtctactactgcgcccagaacctcgaaattccgaggacctttggacaaggtactaaagtggaaatcaagtgataa >Anti-EpCAM scFv-v4 aa  (SEQ ID NO: 466)DIVMTQSPLSLPVTPGEPASISCRSTKSLLHSDGITYLYWYLQKPGQSPQLLIYQLSNLASGVPDRFSSSG                                *                      *SGTDFTLKISRVEAEDEGVYYCAQNLEIPRTFGQGTKVEIKRTGGGGSGGGGSGGGGSGGGGSGQVQLVQSGSELKKPGESVKVSCKASGYTFTNYGMNWVRQAPGQGLKWMGWINTYTGESTYADDFKGRFAFSLDTSASTAYLQISSLKAEDTAVYFCARFAIKGDYWGQGTLVTVSS** >Anti-EpCAM scFv-v4 nt (SEQ ID NO: 467)gatatcgtgatgacacagtcacccctgtcgctccctgtgactcccggagagcctgcgtccatctcgtgccggtcaacaaagtccctgctgcacagcgacggaattacgtatttgtactggtatttgcaaaaacccggacagagcccgcaattgttgatctaccagctctcaaaccttgcgagcggggtcccggatcgctttagctcgtcggggtcaggaactgacttcacactcaaaatctcaagggtcgaggccgaggatgaaggtgtctattactgcgctcagaatctcgaaatcccgcggacctttggtcagggtacgaaggtagagatcaagcgaacgggaggcggtgggtccggtggcggtggttccggcggaggtggttcgggaggaggaggtagcggtcaggtgcagcttgtccagtcgggatcggagctcaagaagccaggagagtcggtgaaagtatcgtgtaaagcctcggggtacacattcacgaactacggcatgaattgggtgagacaagctcccgggcagggcttgaaatggatgggatggattaacacctacacaggggaatccacatatgcggacgacttcaaggggaggttcgcgttttcacttgatacttcagcgagcacggcgtatctccaaatttcgtcgcttaaggcagaagataccgcagtatacttttgcgccagattcgcgattaaaggggactattggggacaggggacccttgtcacggtgtcatcatgataa >Anti-EpCAM scFv-v5 aa  (SEQ ID NO: 468)DIVMTQSPLSLPVTPGEPASISCRSTKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLKISRVEAEDEGVYYCAQNLEIPRTFGGGTKLEIKRTGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGESVKISCKASGYTFTNYGMNWVRQAPGQGLKWMGWINTYTGESTYADDFKGRFAFSLDTSASTAYLQLSSLRSEDTAVYFCARFAIKGDYWGQGTLVTVSS** >Anti-EpCAM scFv-v5 nt  (SEQ ID NO: 469)gatatcgtgatgacacagtcacccctgtcgctccctgtgactcccggagagcctgcgtccatctcgtgccggtcaacaaagtccctgctgcacagcaatggaattacgtatttgtactggtatttgcaaaaacccggacagagcccgcaattgttgatctaccagatgtcaaaccttgcgagcggggtcccggatcgctttagctcgtcggggtcaggaactgacttcacactcaaaatctcaagggtcgaggccgaggatgaaggtgtctattactgcgctcagaatctcgaaatcccgcggacctttggtggaggtacgaagctggagatcaagcgaacgggaggcggtgggtccggtggcggtggttccggcggaggtggttcgggaggaggaggtagccaggtgcagcttgtccagtcgggagcagaggtaaagaagccaggagagtcggtgaaaatctcgtgtaaagcctcggggtacacattcacgaactacggcatgaattgggtgagacaagctcccgggcagggcttgaaatggatgggatggattaacacctacacaggggaatccacatatgcggacgacttcaaggggaggttcgcgttttcacttgatacttcagcgagcacggcgtatctccaactctcgtcgcttcgctccgaagataccgcagtatacttttgcgccagattcgcgattaaaggggactattggggacaggggacccttgtcacggtgtcatcatgataa >Anti-EpCAM scFv-v6 aa  (SEQ ID NO: 470)DIVMTQSPLSLPVTPGEPASISCRSTKSLLHSDGITYLYWYLQKPGQSPQLLIYQLSNLASGVPDRFSSSG                                *                      *SGTDFTLKISRVEAEDEGVYYCAQNLEIPRTFGGGTKLEIKRTGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGESVKISCKASGYTFTNYGMNWVRQAPGQGLKWMGWINTYTGESTYADDFKGRFAFSLDTSASTAYLQLSSLRSEDTAVYFCARFAIKGDYWGQGTLVTVSS** >Anti-EpCAM scFv-v6 nt (SEQ ID NO: 471)gatatcgtgatgacacagtcacccctgtcgctccctgtgactcccggagagcctgcgtccatctcgtgccggtcaacaaagtccctgctgcacagcgacggaattacgtatttgtactggtatttgcaaaaacccggacagagcccgcaattgttgatctaccagctctcaaaccttgcgagcggggtcccggatcgctttagctcgtcggggtcaggaactgacttcacactcaaaatctcaagggtcgaggccgaggatgaaggtgtctattactgcgctcagaatctcgaaatcccgcggacctttggtggaggtacgaagctggagatcaagcgaacgggaggcggtgggtccggtggcggtggttccggcggaggtggttcgggaggaggaggtagccaggtgcagcttgtccagtcgggagcagaggtaaagaagccaggagagtcggtgaaaatctcgtgtaaagcctcggggtacacattcacgaactacggcatgaattgggtgagacaagctcccgggcagggcttgaaatggatgggatggattaacacctacacaggggaatccacatatgcggacgacttcaaggggaggttcgcgttttcacttgatacttcagcgagcacggcgtatctccaactgtcgtcgcttcgctccgaagataccgcagtatacttttgcgccagattcgcgattaaaggggactattggggacaggggacccttgtcacggtgtcatcatgataa >Anti-EpCAM scFv-v7 aa  (SEQ ID NO: 472)DIVMTQSPLSLPVTPGEPASISCRSTKSLLHSDGITYLYWYLQKPGQSPQLLIYQLSNLASGVPDRFSSSGSGTDFTLKISRVEAEDEGVYYCAQNLEIPRTFGCGTKLEIKRTGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGESVKISCKASGYTFTNYGMNWVRQAPGQCLKWMGWINTYTGESTYADDFKGRFAFSLDTSASTAYLQLSSLRSEDTAVYFCARFAIKGDYWGQGTLVTVSS** >Anti-EpCAM scFv-v7 nt (SEQ ID NO: 473)gatatcgtgatgacacagtcacccctgtcgctccctgtgactcccggagagcctgcgtccatctcgtgccggtcaacaaagtccctgctgcacagcgacggaattacgtatttgtactggtatttgcaaaaacccggacagagcccgcaattgttgatctaccagctctcaaaccttgcgagcggggtcccggatcgctttagctcgtcggggtcaggaactgacttcacactcaaaatctcaagggtcgaggccgaggatgaaggtgtctattactgcgctcagaatctcgaaatcccgcggacctttggttgcggtacgaagctggagatcaagcgaacgggaggcggtgggtccggtggcggtggttccggcggaggtggttcgggaggaggaggtagccaggtgcagcttgtccagtcgggagcagaggtaaagaagccaggagagtcggtgaaaatctcgtgtaaagcctcggggtacacattcacgaactacggcatgaattgggtgagacaagctcccgggcagtgtttgaaatggatgggatggattaacacctacacaggggaatccacatatgcggacgacttcaaggggaggttcgcgttttcacttgatacttcagcgagcacggcgtatctccaactgtcgtcgcttcgctccgaagataccgcagtatacttttgcgccagattcgcgattaaaggggactattggggacaggggacccttgtcacggtgtcatcatgataa >Anti-EpCAM scFv-v8 aa  (SEQ ID NO: 474)QVQLVQSGAEVKKPGESVKISCKASGYTFTNYGMNWVRQAPGQGLKWMGWINTYTGESTYADDFKGRFAFSLDTSASTAYLQLSSLRSEDTAVYFCARFAIKGDYWGQGTLVTVSSASTGGGGSGGGGSGGGGSGGGGSDIVMTQSPLSLPVTPGEPASISCRSTKSLLHSDGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGT                             *DFTLKISRVEAEDEGVYYCAQNLEIPRTFGGGTKLEIKRT** >Anti-EpCAM scFv-v8 nt (SEQ ID NO: 475)caggtgcagctcgtccagtcaggggcggaggtcaagaaaccaggagagtcagtaaagatctcgtgcaaagcgtcgggatatacctttacaaactacggcatgaattgggtgcgacaagcacccggccagggcctgaagtggatggggtggatcaatacatatactggggagtccacttatgccgacgacttcaagggaaggtttgccttctccctggatacgtcggcgtcgaccgcttatttgcagttgagctcgctgaggtcggaagatacagcagtgtacttctgcgctcgcttcgcaatcaaaggggattactggggtcaggggacgcttgtaaccgtgtcctcagcgtcgacgggtggtggtgggtcgggaggtggtggtagcggaggtggagggtcgggtggaggcggatcagatattgtgatgacacaatcgccgctctcactgcccgtaacgcccggagagcccgcgtcaatttcatgtcggtcgacaaagtcactccttcactcggacgggattacgtacctctattggtatcttcaaaagccgggtcagtcacctcaactcctcatctaccagatgtcgaacttggcatcaggggtccctgatcgcttctcgtccagcgggtccgggacggactttaccttgaaaatctcaagagtggaggccgaggacgaaggggtctactactgtgcccagaaccttgaaattccgcggacgtttggaggagggacaaagctggagatcaaaagaacttgataa >Anti-EpCAM scFv-v9 aa  (SEQ ID NO: 476)QVQLQQSGGGLVKPGESVKISCAASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGESTYADDFKGRFTESLETSASAAYLQINSLRPEDTAVYFCARFAIKGDYWGQGTTVTVSSASTGGGGSGGGGSGGGGSGGGGSGDIVMTQSPSSLSASVGDKATITCRSTKSLLHSNGITYLYWYLQKPGKAPKLLIYQMSNLASGVPDRFSSSGSGTEFTLTISSVQPEDEGTYYCAQNLEIPRTFGQGTKLEIK** >Anti-EpCAM scFv-v9 nt (SEQ ID NO: 477)caggtacaactccagcaatcaggaggcgggcttgtaaaacccggggagtcggtgaagatcagctgtgcagcatcagggtacacattcacaaactatggcatgaactgggtcaaacaggcgccagggaaggggctgaaatggatgggctggatcaatacgtacactggtgagagcacatacgcggatgatttcaaagggcggtttacgttttcactcgaaacgtcggcgtccgccgcatatcttcaaatcaattccttgaggccggaggacacagcagtctacttctgcgcccgatttgccattaagggtgattattggggccagggaaccacggtgactgtcagctccgcttcgacaggaggaggagggagcggtggaggaggatcgggaggtggagggtcgggtggtggtgggtcaggagacattgtgatgacccagtcgccctcgtcgctttcagcgagcgtcggggacaaggcgaccattacttgtcgctcaactaagtcgttgctgcactccaacgggatcacgtacctctattggtatctccaaaaacctggtaaagcgcctaagctcctgatctaccagatgtccaatttggcgtcgggagtaccggacagattttcaagctccggatcagggactgagttcacacttacgattagctccgtgcagcccgaggatgaagggacctattactgcgcccagaacttggaaatcccgagaaccttcggacagggtacgaagctggaaatcaagtgataa >Anti-EpCAM scFv-v10 aa  (SEQ ID NO: 478)QVQLQQSGGGLVKPGESVKISCAASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGESTYADDFKGRFTESLETSASAAYLQINSLRPEDTAVYFCARFAIKGDYWGQGTTVTVSSASTGGGGSGGGGSGGGGSGGGGSGDIVMTQSPSSLSASVGDKATITCRSTKSLLHSDGITYLYWYLQKPGKAPKLLIYQLSNLASGVPDRFSSSGSG                              *                      *TEFTLTISSVQPEDEGTYYCAQNLEIPRTFGQGTKLEIK** >Anti-EpCAM scFv-v10 nt (SEQ ID NO: 479)caggtccagttgcaacagtccggaggtgggcttgtgaaacccggggagtcggtgaagatctcatgcgcggcttcggggtacacgttcacaaactacgggatgaattgggtcaagcaggcacccgggaaggggttgaaatggatgggttggatcaatacatacactggggagagcacgtatgcagatgacttcaaaggacgctttaccttcagcctcgaaacgagcgcctcagccgcatacctccaaatcaatagcctccggccagaagatactgcggtgtatttctgcgctaggttcgccattaaaggggactattggggacaggggacgacggtgacggtatcatcagcgtcgactggtggaggcggctccggaggaggtgggtccggcggtggcgggtcaggtggtggtggctcgggagatattgtaatgacacagtccccgtcctccctctccgcgagcgtgggagataaggccacgattacatgtcgaagcaccaaatcgctccttcactcggacggaatcacatatttgtattggtacttgcaaaaaccggggaaggcgcctaagctgctgatctaccagctttcgaacctggcgtcaggggtccctgaccggttttcgtcgtcgggaagcgggaccgagtttacacttacgatctcgtcagtacagcccgaggacgaaggaacatactattgcgcccagaacttggagattccgagaacttttggccagggaaccaagctcgaaatcaagtgataa >Anti-EpCAM scFv-v11 aa  (SEQ ID NO: 480)DIVMTQSPSSLSASVGDKATITCRSTKSLLHSNGITYLYWYLQKPGKAPKLLIYQMSNLASGVPDRFSSSGSGTEFTLTISSVQPEDEGTYYCAQNLEIPRTFGQGTKLEIKRTGGGGSGGGGSGGGGSGGGGSGQVQLQQSGGGLVKPGESVKISCAASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGESTYADDFKGRFTESLETSASAAYLQINSLRPEDTAVYFCARFAIKGDYWGQGTTVTVSS** >Anti-EpCAM scFv-v11 nt (SEQ ID NO: 481)gacatcgtcatgacccagtcaccgtcctcactgtcggcgtcggtgggtgataaggccacaattacatgccgcagcacgaaatcactgctccactccaatgggattacatatctctattggtatctccaaaaacccggaaaggcacctaagttgctgatctaccagatgtcgaacttggcatcgggagtacccgataggttctcgtcgtcgggaagcggcacggagttcacgctcaccatttcctcagtccagccggaggacgaaggaacttactactgcgctcagaatcttgaaatcccgcgcacatttggacaagggacgaaacttgaaatcaagcgaactggaggaggtgggtcaggcggaggtgggagcggcggaggcggatcgggtggtggagggtcgggacaggtacagttgcagcagtcgggaggcgggctggtaaaacctggtgaaagcgtcaagatctcatgtgcagcctcagggtatacgttcaccaattacgggatgaactgggtgaagcaggcgccagggaaaggtcttaagtggatgggatggatcaacacttacacgggagagtccacatacgcggatgactttaaggggcggttcacgttttcgttggagacttcagcgtccgctgcctacctccaaatcaactcccttagacccgaggacacagcggtctatttctgtgcgcggtttgccattaaaggtgattattggggacagggtacgactgtgaccgtgtccagctgataa >Anti-EpCAM scFv-v12 aa  (SEQ ID NO: 482)DIVMTQSPSSLSASVGDKATITCRSTKSLLHSDGITYLYWYLQKPGKAPKLLIYQLSNLASGVPDRFSSSGSGTEFTLTISSVQPEDEGTYYCAQNLEIPRTFGQGTKLEIKRTGGGGSGGGGSGGGGSGGGGSGQVQLQQSGGGLVKPGESVKISCAASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGESTYADDFKGRFTESLETSASAAYLQINSLRPEDTAVYFCARFAIKGDYWGQGTTVTVSS >Anti-EpCAM scFv-v12 nt (SEQ ID NO: 483)gacatcgtcatgacccagtcaccgtcctcactgtcggcgtcggtgggtgataaggccacaattacatgccgcagcacgaaatcactgctccactccgacgggattacatatctctattggtatctccaaaaacccggaaaggcacctaagttgctgatctaccagttgtcgaacttggcatcgggagtacccgataggttctcgtcgtcgggaagcggcacggagttcacgctcaccatttcctcagtccagccggaggacgaaggaacttactactgcgctcagaatcttgaaatcccgcgcacatttggacaagggacgaaacttgaaatcaagcgaactggaggaggtgggtcaggcggaggtgggagcggcggaggcggatcgggtggtggagggtcgggacaggtacagttgcagcagtcgggaggcgggctggtaaaacctggtgaaagcgtcaagatctcatgtgcagcctcagggtatacgttcaccaattacgggatgaactgggtgaagcaggcgccagggaaaggtcttaagtggatgggatggatcaacacttacacgggagagtccacatacgcggatgactttaaggggcggttcacgttttcgttggagacttcagcgtccgctgcctacctccaaatcaactcccttagacccgaggacacagcggtctatttctgtgcgcggtttgccattaaaggtgattattggggacagggtacgactgtgaccgtgtccagctgataa >Anti-EpCAM scFv-v13 aa  (SEQ ID NO: 484)DIVMTQSPSSLSASVGDKATITCRSTKSLLHSNGITYLYWYLQKPGKAPKLLIYQMSNLASGVPDRFSSSGSGTEFTLTISSVQPEDEGTYYCAQNLEIPRTFGQGTKLEIKRTPSHNSHQVPSAGGPTANSGTSGSQVQLQQSGGGLVKPGESVKISCAASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGESTYADDFKGRFTFSLETSASAAYLQINSLRPEDTAVYFCARFAIKGDYWGQGTTVTVSS >Anti-EpCAM scFv-v13 nt (SEQ ID NO: 485)gacatcgtcatgacccagtcaccgtcctcactgtcggcgtcggtgggtgataaggccacaattacatgccgcagcacgaaatcactgctccactccaatgggattacatatctctattggtatctccaaaaacccggaaaggcacctaagttgctgatctaccagatgtcgaacttggcatcgggagtacccgataggttctcgtcgtcgggaagcggcacggagttcacgctcaccatttcctcagtccagccggaggacgaaggaacttactactgcgctcagaatcttgaaatcccgcgcacatttggacaagggacgaaacttgaaatcaagcgaactccgtcccacaacagccatcaagtgccctcggcgggagggcccaccgccaattcggggacatcagggagccaggtacagttgcagcagtcgggaggcgggctggtaaaacctggtgaaagcgtcaagatctcatgtgcagcctcagggtatacgttcaccaattacgggatgaactgggtgaagcaggcgccagggaaaggtcttaagtggatgggatggatcaacacttacacgggagagtccacatacgcggatgactttaaggggcggttcacgttttcgttggagacttcagcgtccgctgcctacctccaaatcaactcccttagacccgaggacacagcggtctatttctgtgcgcggtttgccattaaaggtgattattggggacagggtacgactgtgaccgtgtccagctgataa >Murine anti-epCAM-LC (SEQ ID NO: 486)DIVMTQSAFS NPVTLGTSAS ISCRSTKSLL HSNGITYLYW YLQKPGQSPQ LLIYQMSNLASGVPDRFSSS GSGTDFTLRI SRVEAEDVGV YYCAQNLEIP RTFGGGTKLE IK >Murine anti-epCAM-HC(SEQ ID NO: 487)QVQLQQSGPE LKKPGETVKI SCKASGYTFT NYGMNWVKQA PGRGLKWMGW INTYTGESTYADDFKGRFAF SLETSASAAY LQINNLKNED TATYFCARFA IKGDYWGQGT TLTVSS >Humanized MOC31 from WO 2000/061635 (SEQ ID NO: 488)DIQMTQSPSSLSASVGDRVTITCRSTKSLLHSNGITYLYWYQQKPGKAPKLLIYQMSNLASGVPSRFSSSGSGTDFTLTISSLQPEDFATYYCAQNLEIPRTFGQGTKVELKRTPSHNSHQVPSAGGPTANSGTSGSEVQLVQSGPGLVQPGGSVRISCAASGYTFTNYGMNWVKQAPGKGLEWMGWINTYTGESTYADSFKGRFTFSLDTSASAAYLQINSLRAEDTAVYYCARFAIKGDYWGQGTLLTVSS >TFcBA large chain complete(SEQ ID NO: 489)QLQLQESGPGLVKPSETLSLTCTVSGGSISSSVYYWSWIRQPPGKGLEWIGVIYPSGNTYYSPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARTIYDLFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSKYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSKSCDKTGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSDYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSGECGGGGSGGGGSDIVMTQSPLSLPVTPGEPASISCRSTKSLLHS DGITYLYWYLQKPGQSPQLLIYQLSNLASGVPDRFSSSGSGTDFTLKISRVEAEDEGVYYCAQNLEIPRTFGCGTKLEIKRTGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRSTKSLLHSNGITYLYWYQQKPGKAPKLLIYQMSNLASGVPSRFSSSGSGTDFTLTISSLQPEDFATYYCAQNLEIPRTFGQGTKVELKRTPSHNSHQVPSAGGPTANSGTSGSEVQLVQSGPGLVQPGGSVRISCAASGYTFTNYGMNWVKQAPGKGLEWMGWINTYTGESTYADSFKGRFTFSLDTSASAAYLQINSLRAEDTAVYYCARFAIKGDYWGQGTLLTVSS

Example A-14 TFcBAs

In certain embodiments, bispecific antibodies (anti-cMet/anti-EpCAM) areprovided. In such embodiments, the bispecific antibodies comprise an Fabportion wherein there is a binding site with specificity for c-Met andan scFv portion wherein there is a binding site with specificity forEpCAM.

In a particular embodiment, we introduced certain stabilizing featuresin the scFv: (1) removal of a potential de-amidation (NG) site fromCDRL1 and replaced it with DG; (2) removal of a potential methionineoxidation site from CDRL2 and replaced it with Leucine; (3) introductionof a disulfide bridge to further stabilize the VH-VL interface.

Further Examples Section B Example B-1 Quantitative Flow CytometryAssessment of Cell Surface Expression of Bispecific Antibody Antigens

For measurements of cell surface expression of, e.g., cMet, ErbB1,ErbB2, EpCAM, CD44, and CEA levels, quantitative flow cytometry isperformed using the Quantum Simply Cellular kit (Bangs Laboratories).

Cells are grown in exponential phase using standard cell culture mediacontaining 10% FBS, and are passaged at least twice before the start ofthe experiment. On the day of the experiment, cells are visuallyassessed under a microscope to confirm between 60% and 80% confluence.Cells are detached from the culture plate by addition of 0.05%trypsin-EDTA (Gibco), and once a majority of cells are detached (asassessed visually by microscope) the trypsin is inactivated using cellculture media containing 10% FBS. The cells are centrifuged at 500 g,resuspended in flow cytometry buffer (2% FBS+0.1% sodium azide in PBS),and seeded at a density of 50,000 cells per well in a 96-well plate (BDBiosciences, catalog #62406-015).

In a separate 96-well plate, 2 drops of Quantum Simply Cellularanti-mouse IgG coated beads (Bangs Laboratories, catalog #815) oranti-human IgG coated beads (Bangs Laboratories, catalog #816) are addedper well. Each bead kit contains 5 bead populations (1 blank and 4 beadswith increasing levels of Fc-specific capture antibody). Each coatedpopulation binds a specific number of monoclonal antibodies of theappropriate species (the “ABC” value), and thus serves as a standardcurve for quantification when beads are labeled to saturation with thesame monoclonal antibody that is used to label cell surface protein.

Antibodies against the cell surface targets are given in Table B-1. Forantibodies against c-Met, ErbB1, ErbB2, and CEA, conjugation with AlexaFluor 647 (Life Technologies, catalog #A-20006) is done according tomanufacturer's instructions. Antibodies against EpCAM and CD44 areavailable pre-conjugated with APC.

TABLE B-1 Target Antibody Source c-Met 224G11-TH7-Hz3 United Statespatent application 2011/0097262: SEQ ID NO 4 (V_(H) domain), 10 (V_(L)domain), and 28 (hinge region) ErbB1 cetuximab Bristol-Myers Squibb/EliLilly ErbB2 trastuzumab Genentech EpCAM EpCAM APC clone BD Biosciences,catalog #347200 EBA-1 CD44 clone G44-26 APC BD Biosciences, catalog#559942 CEA clone B6.2 BD Biosciences, catalog #551355

The appropriate fluorophore-conjugated antibody is added to the cellsand the beads (200 nM antibody concentration in 80 μl of flow cytometrybuffer). Antibody is allowed to incubate for 30 minutes at 4° C. Theplates are centrifuged and washed twice with 100 μL of ice-cold flowcytometry buffer. After the last wash, the cells and beads arecentrifuged and resuspended in 100 μL of ice-cold flow cytometry bufferand read using the appropriate fluorescence filter on a flow cytometer(BD FACSCanto). Channel values for the bead populations are recorded inthe bead lot-specific QuickCal template provided in the Quantum SimplyCellular kit. A regression is performed that relates fluorescence signalto the beads' ABC values. ABC values are assigned to stained cellsamples using this standard curve. If monovalent antibody-to-cellsurface receptor binding is presumed, then the ABC value equals thenumber of surface receptors.

Table B-2 lists cell surface expression levels in a panel of cell linesderived from colorectal, ovarian, lung, breast, brain, gastric,prostate, and pancreatic cancers measured using the above protocol.Table B-3 lists the ratio of expression between the various cell surfacetargets and c-Met. It can be seen that EpCAM has the highest medianexpression level of any measured target, and also the highest medianexpression ratio relative to c-Met, supporting the selection of EpCAM asa targeting moiety for potent bispecific antibody binding of c-Met.

In the following 2 Tables, the Tumor Types are indicated as follows:1=colorectal, 2=ovarian, 3=non-small cell lung, 4=renal, 5=breast,6=glioma, 7=triple-negative breast, 8=gastric, 9=epidermoid,10=prostate, and 11=pancreatic. Cell lines indicated are commerciallyavailable, e.g., from ATCC.

TABLE B-2 Tumor c-Met ErbB1 ErbB2 EpCAM CD44 CEA Cell Line Type (#/cell)(#/cell) (#/cell) (#/cell) (#/cell) (#/cell) HCC2998 1 8.6E+04 1.4E+058.6E+04 2.5E+06 5.6E+04 2.2E+05 IGROV-1 2 1.3E+05 1.8E+05 1.5E+059.3E+02 1.3E+04 3.9E+04 SW620 1 8.7E+04 9.9E+03 2.3E+04 1.7E+06 1.2E+033.0E+04 H441 3 3.2E+05 6.6E+05 1.0E+05 2.5E+06 2.4E+02 2.7E+05 ACHN 41.3E+05 7.8E+05 5.0E+04 4.6E+05 6.4E+06 3.6E+04 HT-29 1 9.6E+04 2.2E+058.6E+04 2.5E+06 9.3E+05 6.8E+04 HCC1954 5 1.3E+05 2.9E+05 8.0E+052.4E+06 1.4E+06 4.6E+06 A549 3 7.2E+04 3.2E+05 5.0E+04 2.4E+04 4.8E+063.2E+04 U87 6 3.2E+04 2.0E+05 1.0E+04 4.0E+02 4.9E+06 1.0E+04 HCT-15 12.3E+04 1.4E+05 6.1E+04 2.1E+06 4.0E+03 1.3E+04 MKN45 8 1.7E+05 1.6E+051.0E+05 1.8E+06 3.1E+05 1.3E+07 colo205 1 2.9E+04 1.0E+05 1.5E+052.1E+06 1.7E+05 7.4E+03 U251 6 7.4E+04 2.9E+05 2.9E+04 2.0E+02 4.1E+061.7E+04 HCC827 3 2.1E+05 1.1E+06 9.3E+04 2.1E+06 1.9E+05 8.8E+05 EKVX 34.8E+04 3.6E+05 1.1E+05 1.7E+04 2.3E+06 1.0E+04 H1975 3 8.1E+04 1.5E+056.4E+04 2.1E+05 3.2E+06 3.4E+04 H596 3 5.4E+04 1.7E+05 5.6E+04 2.1E+063.6E+05 1.3E+04 JIMT 7 3.7E+04 4.6E+05 2.8E+05 2.0E+06 1.4E+06 2.6E+04BT20 7 2.5E+04 1.3E+06 4.5E+05 2.3E+06 9.3E+05 6.7E+04 MCF7 5 7.3E+038.7E+03 3.8E+04 2.6E+06 1.7E+05 2.8E+04 Cal51 7 1.6E+04 7.1E+04 3.5E+041.2E+06 2.1E+06 1.4E+04 Calu3 3 7.7E+04 3.2E+05 9.3E+05 2.6E+06 1.6E+062.5E+05 SNU5 8 3.0E+05 2.5E+05 4.6E+05 1.9E+06 5.8E+04 7.2E+04 H1993 36.1E+05 4.0E+05 6.5E+04 2.6E+06 4.9E+05 1.6E+07 A431 9 2.4E+04 1.6E+061.5E+05 1.5E+06 1.1E+06 2.3E+05 SkBr3 5 3.3E+04 3.4E+05 1.2E+06 3.0E+063.3E+03 1.4E+05 NCI- 3 5.8E+04 5.4E+04 1.2E+06 3.9E+06 1.4E+04 1.0E+07H2170 PC3 10 1.2E+05 3.1E+05 6.9E+04 7.9E+04 2.7E+06 6.9E+04 H1299 37.4E+04 3.0E+05 5.6E+04 1.2E+03 4.3E+06 7.4E+04 H1703 3 3.4E+04 5.3E+058.3E+04 1.2E+03 4.7E+02 3.2E+04 MDA-MB 5 9.2E+04 3.5E+05 5.1E+04 3.1E+045.2E+06 6.1E+04 231 BxPC3 11 1.2E+05 9.2E+05 6.6E+04 7.1E+05 5.9E+051.0E+07 Cal120 7 1.3E+05 8.4E+05 1.1E+05 1.1E+05 2.1E+07 1.0E+06 H2347 35.4E+05 9.6E+05 2.3E+05 9.2E+06 7.0E+05 6.0E+06 OVCAR8 2 1.1E+05 7.7E+051.0E+05 3.4E+05 1.2E+07 8.2E+05 SkOV3 2 1.6E+05 6.9E+05 2.6E+06 3.2E+051.1E+07 5.8E+05 MDAMB- 5 2.9E+04 5.1E+04 1.4E+06 7.5E+06 1.5E+03 4.2E+05453 BT474-M3 5 4.2E+04 2.1E+05 2.7E+06 9.2E+06 1.4E+03 4.2E+07 BT549 52.3E+05 1.8E+06 1.3E+05 7.4E+03 2.3E+07 1.5E+06 OVCAR3 2 1.3E+05 9.5E+051.7E+05 9.2E+06 4.9E+04 1.0E+06 OVCAR-4 2 4.9E+04 3.3E+05 1.1E+052.6E+06 1.7E+05 3.1E+05 OVCAR-5 2 5.5E+04 8.0E+05 9.1E+04 2.0E+069.9E+04 3.5E+06 H522 3 3.1E+04 5.4E+04 1.1E+05 2.6E+03 1.1E+03 2.8E+05CaOV3 2 1.0E+05 6.3E+05 1.0E+05 2.6E+06 1.7E+05 4.9E+05 SW-626 2 2.0E+054.2E+05 1.0E+05 2.6E+06 1.2E+05 4.5E+05 HCT116 1 7.5E+04 2.1E+05 5.5E+042.6E+06 5.3E+05 2.1E+05 HCT-8 1 4.3E+04 2.2E+05 6.2E+04 2.6E+06 6.7E+042.4E+05 KM-12 1 4.0E+04 8.5E+04 6.0E+04 2.6E+06 7.2E+05 4.1E+05 H-23 37.4E+04 2.8E+05 5.2E+04 1.6E+04 1.7E+06 5.5E+05 OV-90 2 6.6E+04 9.8E+046.4E+04 2.4E+06 3.2E+06 2.2E+06 OVCA- 2 1.7E+05 6.9E+05 8.4E+04 2.4E+069.6E+05 5.6E+05 433 TOV- 2 2.1E+04 3.2E+04 7.7E+04 2.4E+03 0.0E+001.6E+05 112D LoVo 1 3.8E+04 1.6E+05 4.9E+04 2.4E+06 3.4E+05 3.0E+05LS174T 1 3.4E+04 1.1E+05 7.3E+04 2.4E+06 4.6E+05 5.1E+05 ADRr 5 4.2E+042.9E+05 6.1E+04 2.0E+05 2.9E+06 2.4E+05 DU145 10 4.0E+04 3.9E+05 5.8E+044.5E+05 2.0E+06 3.4E+05 Median 7.4E+04 3.0E+05 8.6E+04 2.1E+06 5.9E+052.5E+05

TABLE B-3 Tu- mor ErbB1/ ErbB2/ EpCAM/ CD44/ CEA/ Cell Line Type c-Metc-Met c-Met c-Met c-Met HCC2998 1 1.6 1.0 29.1 0.7 2.6 IGROV-1 2 1.4 1.20.0 0.1 0.3 SW620 1 0.1 0.3 19.5 0.0 0.3 H441 3 2.1 0.3 7.8 0.0 0.8 ACHN4 6.0 0.4 3.5 49.2 0.3 HT-29 1 2.3 0.9 26.0 9.7 0.7 HCC1954 5 2.2 6.218.5 10.8 35.4 A549 3 4.4 0.7 0.3 66.7 0.4 U87 6 6.3 0.3 0.0 153.1 0.3HCT-15 1 6.1 2.7 91.3 0.2 0.6 MKN45 8 0.9 0.6 10.6 1.8 76.5 colo205 13.4 5.2 72.4 5.9 0.3 U251 6 3.9 0.4 0.0 55.4 0.2 HCC827 3 5.2 0.4 10.00.9 4.2 EKVX 3 7.5 2.3 0.4 47.9 0.2 H1975 3 1.9 0.8 2.6 39.5 0.4 H596 33.1 1.0 38.9 6.7 0.2 JIMT 7 12.4 7.6 54.1 37.8 0.7 BT20 7 52.0 18.0 92.037.2 2.7 MCF7 5 1.2 5.2 356.2 23.3 3.8 Cal51 7 4.4 2.2 75.0 131.3 0.9Calu3 3 4.2 12.1 33.8 20.8 3.2 SNU5 8 0.8 1.5 6.3 0.2 0.2 H1993 3 0.70.1 4.3 0.8 26.2 A431 9 66.7 6.3 62.5 45.8 9.6 SkBr3 5 10.3 36.4 90.90.1 4.2 NCI-H2170 3 0.9 20.7 67.2 0.2 172.4 PC3 10 2.6 0.6 0.7 22.5 0.6H1299 3 4.1 0.8 0.0 58.1 1.0 H1703 3 15.6 2.4 0.0 0.0 0.9 MDA-MB 231 53.8 0.6 0.3 56.5 0.7 BxPC3 11 7.7 0.6 5.9 4.9 83.3 Cal120 7 6.5 0.8 0.8161.5 7.7 H2347 3 1.8 0.4 17.0 1.3 11.1 OVCAR8 2 7.0 0.9 3.1 109.1 7.5SkOV3 2 4.3 16.3 2.0 68.8 3.6 MDAMB-453 5 1.8 48.3 258.6 0.1 14.5BT474-M3 5 5.0 64.3 219.0 0.0 1000.0 BT549 5 7.8 0.6 0.0 100.0 6.5OVCAR3 2 7.3 1.3 70.8 0.4 7.7 OVCAR-4 2 6.7 2.2 53.1 3.5 6.3 OVCAR-5 214.5 1.7 36.4 1.8 63.6 H522 3 1.7 3.5 0.1 0.0 9.0 CaOV3 2 6.3 1.0 26.01.7 4.9 SW-626 2 2.1 0.5 13.0 0.6 2.3 HCT116 1 2.8 0.7 34.7 7.1 2.8HCT-8 1 5.1 1.4 60.5 1.6 5.6 KM-12 1 2.1 1.5 65.0 18.0 10.3 H-23 3 3.80.7 0.2 23.0 7.4 OV-90 2 1.5 1.0 36.4 48.5 33.3 OVCA-433 2 4.1 0.5 14.15.6 3.3 TOV-112D 2 1.5 3.7 0.1 0.0 7.6 LoVo 1 4.2 1.3 63.2 8.9 7.9LS174T 1 3.2 2.1 70.6 13.5 15.0 ADRr 5 6.9 1.5 4.8 69.0 5.7 DU145 10 9.81.5 11.3 50.0 8.5 Me- 4.1 1.2 14.1 7.1 3.8 dian ratio

Example B-2 Dose-Response Analysis of Antibody Activity on U-87 MGTumors Implanted into Nude Mice

The in vivo activity of Ab#7 can be further explored in the U-87autocrine HGF model through an experiment testing the response of U-87MG tumors to different doses of antibody. The dose-response evaluationcan proceed according to the protocol described in Example A-12, exceptthat drug is dosed every seven days. The following groups are treated:

(1) PBS control

(2) Ab#7, approximately 24 mg/kg based on individual mouse body weight

(3) Ab#7, approximately 12 mg/kg

(4) Ab#7, approximately 4 mg/kg

(5) Ab#7, approximately 1 mg/kg

Data from a representative experiment conducted using the above protocolare shown in FIG. 32. Treatment with Ab#7 resulted in growth stasis ofsubcutaneously implanted U-87 MG tumors at the 1 mg/kg dosing level, andtumor regression at the 4 mg/kg, 12 mg/kg, and 24 mg/kg dosing levels. Adose level of equal to or higher than 12 mg/kg, given every seven days,had maximal anti-tumor activity in this experiment.

Example B-3 Creation of NCI-H358 Cell Line Variant that Secretes HumanHGF (H358-HGF Cells)

Transfection of a human HGF transgene into NCI-H358 lung cancer cells(ATCC CRL-5807) may be performed using lentiviral particles (GeneCopoeiacatalog #LP-A0820_Lv105-0200) according to the following protocol.NCI-H358 cells grown using standard cell culture medium supplementedwith 10% fetal bovine serum (FBS) are trypsinized, counted, and seededat a density of 10,000 cells per well in a 96-well plate, using a finalvolume of 1004 Three μl of lentiviral particles are added per well. Theplate is spun in a centrifuge at 2000 rpm for 2 hours at 37° C., andthen removed from the centrifuge and placed in a 37° C. incubator for 4days. After 4 days, the media is aspirated and replaced with freshgrowth media without lentivirus. After 24 hours of further incubation,the cells are trypsinized and transferred to a 24-well plate. Aftergrowth to 80% confluence, the cells are again trypsinized andtransferred to a 6-well plate. After growth to 80% confluence, the mediais aspirated and replaced with media containing puromycin selectionantibiotic at a final concentration of 1 μg/ml. Cells are regrown inselection media until 80% confluence, after which the cells aretrypsinized and pooled for expansion and later use.

Example B-4 Luminex-Based Quantification of HGF Secretion in H358-HGFCell Line

The secretion of human HGF from cell lines may be measured using abead-based sandwich immunoassay system (Luminex) coupled to anti-HGFantibody (R&D Systems, catalog #DY294), as described below. Preparationof beads may be performed as follows. Bio-Plex COOH beads (Bio-Rad,catalog #171-506052) are vortexed for 30 seconds, followed by bath-basedsonication for an additional 30 seconds. Place 100 μl of beads into anEppendorf tube and centrifuge at 12,000 RPM for 4 minutes. After removalof the supernatant, add 100 μl of bead wash buffer (Bio-Rad Bio-PlexAmine Coupling Kit, catalog #171-406001). Vortex and sonicate theEppendorf tube for 10 seconds each, followed by additionalcentrifugation at 12,000 RPM for 4 minutes. After removal of thesupernatant, resuspend the bead pellet in 80 μl of bead activationbuffer (Bio-Rad Bio-Plex Amine Coupling Kit, catalog #171-406001).Vortex and sonicate the Eppendorf tube for 30 seconds each.

Immediately before use prepare 50 mg/ml solutions of both EDCcarboxyl/amine crosslinker (Thermo Scientific, catalog #22980) andSulfo-NHS ester conversion reagent (Thermo Scientific, catalog #24510).Add 10 μl of EDC solution to the Eppendorf tube, followed by 10 μl ofsulfo-NHS solution. Vortex the Eppendorf for 30 seconds, cover the tubewith aluminum foil to protect from light, and agitate the beads in arotator for 20 minutes at room temperature. Add 150 μl of PBS, vortexthe activated beads for 10 seconds, and then centrifuge at 12,000 RPMfor 4 minutes. Remove the supernatant, and wash twice with 500 μl ofPBS. Resuspend the activated beads in 100 μl antibody (1 mg/ml),followed by rotation for 2 hours at room temperature protected fromlight. A magnet is used to pellet the beads, so that the buffer can beremoved carefully without disrupting the beads. Next, resuspend thebeads in 1 ml of PBS-TBN (PBS, 0.1% BSA, 0.02% Tween-20, 0.05% Azide, pH7.4) so that they can be stored at 4° C. in the dark until use.

Prior to use of the beads, centrifuge at 12,000 rpm for 4 minutes.Remove and discard the supernatant and wash with PBS one time andreplace the supernatant with 250 μl blocking buffer (1% BSA in PBS).Vortex the beads gently for 15 seconds and then agitate using a rotatorat room temperature for 30 minutes protected from light. Preparestandards for HGF using two-fold serial dilutions of recombinant proteinstarting at 5 ng/ml (R&D Systems, catalog #DY294). Add recombinantstandard solutions and cell line supernatant to the beads and incubateovernight at 4° C. on a shaker protected from light. Dilute thedetection antibody (R&D Systems, catalog #DY294) in streptavidin-PE inPBS+1% BSA to achieve a working solution of 0.001 mg/ml. Allow the beadsto precipitate for 5 minutes on a magnetic plate at 4° C. Hold the platecontaining the samples on the magnet firmly and invert the plate into asink and gently blot to remove excess moisture. Centrifuge the platebriefly and add 200 μl of PBS containing 1% BSA to each well. Allow thebeads to pellet on the magnet for 1-2 minutes protected from light.Repeat the above washing steps by holding the plate firmly to the magnetand inverting into the sink and adding PBS containing 1% BSA for a totalof three washes. Add 100 μl of the prepared working solution ofdetection antibody per well. Shake gently for 1 hour protected fromlight. Upon completion of the incubation with detection antibody, repeatthe wash steps a total of three times using PBS containing 1% BSA.Resuspend the samples in 100 μl PBS containing 1% BSA and read on theLuminex FlexMAP3D plate reader. All data is regressed to the standardcurve generated using recombinant protein and then normalized based onthe protein content of the cells used to generate the cell culturesupernatant. Protein concentration for all cells can be performed permanufacturer's recommendation using the Thermo Scientific Pierce BCAProtein Assay (catalog #23225).

Using the above protocol, concentration of HGF in the supernatant ofcell culture media for H358-HGF and U-87 MG cells was measured andsubsequently normalized to the protein concentration of cells. TheH358-HGF cells demonstrated a high level of HGF secretion relative tothat of U-87 MG cells that natively express autocrine HGF (FIG. 33),whereas the parental and mock-transfected H358 cells did not expressdetectable HGF.

Example B-5 Bispecific Antibody Activity in EpCAM-Expressing Tumor CellLines Implanted into Nude Mice

To explore the role of tumor EpCAM expression in promoting the in vivoactivity of Ab#7, the molecule can be evaluated in c-Met driven tumormodels expressing EpCAM at levels significantly higher than c-Met. ForHGF-ligand dependent tumor models, cell lines require autocrinesecretion of human HGF, as it is known that mouse HGF does not activatehuman c-Met.

HCC827 cells transfected with human HGF (HCC827-HGF cells), along withmock-transfected HCC827 cells, were obtained from Dr. Jeffrey A.Engelman and were created according to the protocol described in Okamotoet al., Mol. Cancer Ther. 9(10):2785-92. NCI-H358 cells transfected withhuman HGF (H358-HGF cells), and mock-transfected control cells, werecreated as per the protocol of Example B-3.

Quantitative flow cytometry measurements of HCC827 cell line variantsdemonstrated that the level of c-Met decreased in the HGF-transfectedcells relative to mock-transfected parental cells (1.2×10⁵/cell versus3.8×10⁵/cell), but that the level of EpCAM was unaffected (2.2×10⁶/cellversus 2.2×10⁶/cell).

Quantitative flow cytometry measurements of H358 cell line variantsdemonstrated that the level of c-Met remained constant in theHGF-transfected cells relative to mock-transfected parental cells(4.6×10⁵/cell versus 4.5×10⁵/cell), but that the level of EpCAM wasincreased (4.9×10⁷/cell versus 3.4×10⁷/cell).

For in vivo studies, cells are cultured in T75 flasks under a humidifiedatmosphere of 5% CO₂ at 37° C. in RPMI 1640 medium (Sigma, St. Louis,Mo.) supplemented with 10% fetal bovine serum (FBS). Cells are harvestedby exposure to 0.25% trypsin. Cells are washed twice inphosphate-buffered saline (PBS) and resuspended in growth factor reducedMatrigel (BD Biosciences) at a 1:1 ratio in PBS. The cells are thencounted with Trypan Blue (Life Technologies, catalog #15250-061) andmice are not implanted if the viability is <90%.

The in vivo activity of Ab#7 and OA-5D5 in these tumor models can beevaluated essentially as follows: Six-to-seven-week-old female Nu/Numice (Charles River Laboratories, Wilmington, Mass.) are injectedsubcutaneously with either 5×10⁶ H358-HGF cells/mouse or 5×10⁶cells/mice HCC827-HGF using an injection volume of 200 μl PBS. Upontumor take after injection, initial tumor volumes are measured using thefollowing formula: (n/6)*L*W². The mice are sorted by weight torandomize the initial tumor volume range of 200±50 mm³, at which timethe animals are sorted into treatment groups of eight animals per group.Mice are treated with PBS control, bispecific antibody, or OA-5D5 byintraperitoneal injection every 7 days.

The doses of the bispecific antibody correspond to approximately 1mg/kg, 4 mg/kg, 12 mg/kg, and 24 mg/kg based on body weight, while thedose of OA-5D5 is approximately 10 mg/kg (an equal molar level with the12 mg/kg bispecific antibody dose). Tumor measurements and body weightsare determined twice weekly throughout the study. The tumor volume isdetermined by measuring in two directions with fine calipers andcalculated using the following formula: tumor volume=((π/6)*L*W²).Plotted data of tumor size represents mean and standard error of themean for each measurement. Upon study completion, mice are euthanizedand tumors from all animals are excised, flash frozen in liquid N₂, andstored in a −80° C. freezer.

Data from a representative experiment using ligand-dependent HCC827-HGFcells in the above protocol are shown in FIG. 34. At the equimolardosing levels of 10 mg/kg and 12 mg/kg OA-5D5 and Ab#7, respectively,the bispecific antibody had improved activity over OA-5D5. Additionally,the dose-response relationship of Ab#7 demonstrated that a dose level ofequal to or higher than 12 mg/kg had maximal anti-tumor activity in thisexperiment. This result was in agreement to that observed in the U-87 MGmodel described in Example B-2.

Data from a representative experiment using ligand-dependent H358-HGFcells in the above protocol are shown in FIG. 35. At the equimolardosing levels of 10 mg/kg and 12 mg/kg OA-5D5 and Ab#7, respectively,the bispecific antibody caused tumor regression, whereas OA-5D5 did notmeaningfully slow tumor growth relative to control.

Example B-6 Immunoblot Analysis of c-Met and EpCAM Degradation in CellLysates Mediated by Bispecific Antibodies

The ability of antibodies to cause degradation of cellular c-Met andEpCAM can be assessed in cell lysate samples using immunoblotting asfollows. A549, NCI-H441, and NCI-H2170 cells are grown to 70-80 percentconfluence in 12-well plates with RPMI Media (GIBCO, catalog#1187-085)containing 10% Fetal Bovine Serum (Tissue Culture Biologicals,catalog#101H1), 2 mM L-Glutamine (GIBCO, catalog#25030-081), andpenicillin/streptomycin (CeliGro, catalog#30-002-CI).

Cells are then incubated with 100 nM of OA-5D5 or Ab#7 for 24 hours at37° C., 5% CO₂. Upon completion of incubation, the cells are washed 2×with cold PBS and lysed in 200 μL per well of M-PER solution (VWRInternational, catalog #PI78505)+150 mM NaCl+protease and phosphataseinhibitor (cOmplete Protease Inhibitor Cocktail Tablet provided in EASYpacks, Roche Diagnostics Corp, catalog #4693124001; PhosSTOP PhosphataseInhibitor Cocktail Tablets, Roche Diagnostics Corp, catalog#4906837001).

Protein concentration is estimated using the Thermo Scientific PierceBCA Protein Assay (catalog #23225) following the recommended protocol.Briefly, equal volumes of protein standards and cell lysates areincubated at a 1:20 ratio for 30 minutes at 37° C. then absorbance ismeasured at 562 nm. The protein concentration is estimated by regressingthe standards using a linear fit and linearity of serial dilutions ofthe cell lysates.

For Western blot analysis, 10 μg of protein for each cell line andcondition is separated via SDS-PAGE using a 10% gel. The proteins arethen transferred to a PVDF membrane and blotted using total c-Metantibody (Cell Signaling, catalog #8198, 1:100 dilution), EpCAM antibody(Cell Signaling, catalog #2929, 1:100 dilution), or β-actin antibody(Cell Signaling, catalog #4970, 1:100 dilution), all used according tothe manufacturer's recommendations. Goat anti-mouse IRDye 680 and goatanti-rabbit IRDye 800 (LI-COR Biosciences) were used as secondaryantibodies. Immunoblots are imaged and protein levels were quantifiedand normalized to beta-actin levels using an Odyssey imager (LI-CORBiosciences).

As shown in FIG. 36, A549 cells (A), NCI-H441 (B and D), and NCI-H2170cells (C and E) were grown and incubated with 100 nM of OA-5D5 or Ab#7as described above and the relative protein concentration of c-Met (FIG.36, A-C) or EpCAM (D-E) was measured. EpCAM was not detectable in theA549 cells in this assay. As can be seen in FIGS. 19B and 19C,incubation with Ab#7, but not OA-5D5, caused degradation of c-Met inNCI-H441 and HCl-H2170 cells. EpCAM was not degraded in the NCI-H441 orNCI-H2170 cells, regardless of antibody treatment.

Example B-7 Immunoblot Analysis of c-Met and EpCAM Degradation inXenograft Tumors Mediated by Bispecific Antibodies

The ability of antibodies to cause degradation of cellular c-Met andEpCAM can be assessed in xenograft tumor samples using immunoblotting.To do this, tumor samples from end of study tumors from the experimentsdescribed in Example B-3 are surgically obtained and pulverized usingthe CryoPrep system (Covaris) according to manufacturer's instructionsby placing samples in tissue tube pulverizing bags, flash freezing usingliquid nitrogen, and dry pulverization at impact level 3. The freeze andpulverization steps are repeated 3-5 times depending on tissue size.Pulverized samples in tissue bags are inverted and flick-transferred tocryogenic vials (Corning) then weighed for final tumor weight of ˜1-2 mgper sample. The weighed samples are then lysed for 10 minutes on ice intissue protein extraction reagent, TPER (Thermo Scientific) withsupplemented with fresh protease and phosphatase inhibitor cocktailtablet (ROCHE) for a final concentration of ˜200 mg/ml per sample.Further pre-clearing centrifugation (14,000 rpm for 10 minutes) are doneby using QIAshredder (QIAGEN) at 4° C. Centrifuged supernatant isremoved and its total protein concentration estimated using the ThermoScientific Pierce BCA Protein Assay (catalog #23225) per manufacturer'srecommendation. Briefly, equal volumes of protein standards and celllysates are incubated at a 1:20 ratio for 30 minutes at 37° C. thenabsorbance is measured at 562 nm. The protein concentration is estimatedby regressing the standards using a linear fit and linearity of serialdilutions of the cell lysates.

For each immunoblot, an equal amount of total proteins are taken fromindividual tumor lysate samples and combined in their respectivetreatment groups for a total protein amount of 30 mg, which is loadedand run in 1×XT MES running buffer (BIO-RAD) on NuPAGE 4-12% Bis-Tris10-12 well gel (Life Technology). Protein samples on the gel aretransferred to nitrocellulose membrane using the iBlot gel transfersystem (Invitrogen). The membrane is blocked at room temperature for 1hour, probed with various primary antibodies overnight and secondaryantibodies for one hour. All immunoblots are then washed 3× with Trisbuffered Saline with Tween 20 (TTBS, Cell Signaling) Immunoblots areimaged and protein levels were quantified and normalized to beta-actinlevels using an Odyssey imager (LI-COR Biosciences).

Antibodies for use in these studies include: c-Met (D1C2) XP Rabbit mAb#8198, EpCAM (D1B3) Rabbit mAb #2626, β-Actin (13E5) Rabbit mAb #4970(all from Cell Signaling), all are used according to the manufacturer'srecommendations. Goat anti-mouse IRDye 680 and goat anti-rabbit IRDye800 (LI-COR Biosciences) are used as secondary antibodies.

Similar to the results of the in vitro experiment described above inExample B-7, tumors from mice treated with Ab #7, but not mice treatedwith OA-5D5, will show increased degradation of c-Met. Similarly,degradation of EpCAM will not be observed in tumors from mice treatedwith either Ab#7 or OA-5D5.

ADDITIONAL STATEMENTS REGARDING INCORPORATION BY REFERENCE ANDVARIATIONS

All references cited throughout this application, for example patentdocuments including issued or granted patents or equivalents; patentapplication publications; and non-patent literature documents or othersource material; are hereby incorporated by reference herein in theirentireties, as though individually incorporated by reference. Anysequence listing and sequence listing information is considered part ofthe disclosure herewith.

When a Markush group or other grouping of components or items is usedherein, all individual members of the group and all combinations andsubcombinations possible of the group are intended to be individuallyincluded in the disclosure.

As used herein, the singular forms “a”, “an”, and “the” include pluralreference unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andequivalents thereof known to those skilled in the art, and so forth. Aswell, the terms “a” (or “an”), “one or more” and “at least one” can beused interchangeably herein. It is also to be noted that the terms“comprising”, “including”, and “containing” can be used interchangeably.The expression “of any of claims XX-YY” (wherein XX and YY refer toclaim numbers) is intended to provide a multiple dependent claim in thealternative form, and in some embodiments is interchangeable with theexpression “as in any one of claims XX-YY.”

Whenever a range of values is given in the specification, for example, atemperature range, a time range, or a composition, concentration, orother value range, all intermediate ranges and subranges, as well as allindividual values included in the ranges given are intended to beincluded in the disclosure. As used herein, ranges specifically includethe values provided as endpoint values of the range. For example, arange of 1 to 100 specifically includes the end point values of 1 and100. It will be understood that any subranges or individual values in arange or subrange that are included in the description herein can beexcluded from the claims herein.

As used herein, “comprising” is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps. As usedherein, “consisting of” excludes any element, step, or ingredient notspecified in the claim element. As used herein, “consisting essentiallyof” does not exclude materials or steps that do not materially affectthe basic and novel characteristics of the claim. In each instanceherein any of the terms “comprising”, “consisting essentially of” and“consisting of” may be optionally replaced with either of the other twoterms, thus describing alternative aspects of the scope of the subjectmatter. The invention illustratively described herein suitably may bepracticed in the absence of any element or elements, limitation orlimitations which is not specifically disclosed herein.

One of ordinary skill in the art will appreciate that startingmaterials, biological and chemical materials, biological and chemicalreagents, synthetic methods, purification methods, analytical methods,assay methods, and biological methods other than those specificallyexemplified can be employed in the practice of the invention withoutresort to undue experimentation. All art-known functional equivalents,of any such materials and methods are intended to be included in thisdisclosure.

The terms and expressions which have been employed herein are used asterms of description and not of limitation, and there is no intention inthe use of such terms and expressions of excluding any equivalents ofthe features shown and described or portions thereof, but it isrecognized that various modifications are possible within the scope ofthe invention claimed. Thus, it should be understood that althoughaspects of the present invention have been specifically disclosed byvarious embodiments which may include preferred embodiments, exemplaryembodiments and optional features, modifications and variations of theconcepts herein disclosed may be resorted to by those skilled in theart. Such modifications and variations are considered to be within thescope of embodiments of the invention as described and as may be definedby the appended claims.

1-21. (canceled)
 22. A binding agent having a c-Met binding sitecomprising the CDR1, CDR2, and CDR3 of a heavy chain variable regioncomprising the amino acid sequence of positions 1-118 of SEQ ID NO: 410,and the CDR1, CDR2, and CDR3 of a light chain variable region comprisingthe amino acid sequence of positions 1-108 of SEQ ID NO:
 400. 23. Abinding agent having a c-Met binding site, wherein the c-Met bindingsite comprises a heavy chain variable region comprising an amino acidsequence which is at least 90% identical to the amino acid sequence ofpositions 1-118 of SEQ ID NO: 410, and a light chain variable regioncomprising an amino acid sequence which is at least 90% identical to theamino acid sequence of positions 1-108 of SEQ ID NO:
 400. 24. Thebinding agent of claim 22, wherein the c-Met binding site comprises aheavy chain variable region comprising the amino acid sequence ofpositions 1-118 of SEQ ID NO: 410, and a light chain variable regioncomprising the amino acid sequence of positions 1-108 of SEQ ID NO: 400.25. The binding agent of claim 22, which is an antibody.
 26. The bindingagent of claim 25, wherein the antibody is a monoclonal antibody. 27.The binding agent of claim 25, wherein the antibody is an IgG antibody.28. An antibody which competes for binding to c-Met with the bindingagent of claim
 24. 29. An antibody which binds to the same epitope onc-Met as the binding agent of claim
 24. 30. A nucleic acid encoding theheavy and/or light chain variable region of the binding agent of claim22.
 31. A pharmaceutical formulation comprising the binding agent ofclaim 22 and a pharmaceutically acceptable carrier.
 32. A method oftreating cancer comprising administering to a subject in need thereofthe binding agent of claim
 22. 33. A binding agent having a EpCAMbinding site comprising the CDR1, CDR2, and CDR3 of a heavy chainvariable region comprising the amino acid sequence of positions 135-250of SEQ ID NO: 472, and the CDR1, CDR2, and CDR3 of a light chainvariable region comprising the amino acid sequence of positions 1-113 ofSEQ ID NO:
 472. 34. The binding agent of claim 33, wherein the EpCAMbinding site comprises a heavy chain variable region comprising theamino acid sequence of positions 135-250 of SEQ ID NO: 472, and theCDR1, CDR2, and CDR3 of a light chain variable region comprising theamino acid sequence of positions 1-113 of SEQ ID NO:
 472. 35. Thebinding agent of claim 33, which is an antibody.
 36. The binding agentof claim 35, wherein the antibody is a monoclonal antibody.
 37. Thebinding agent of claim 35, wherein the antibody is an IgG antibody. 38.A nucleic acid encoding the heavy and/or light chain variable region ofthe binding agent of claim
 33. 39. A pharmaceutical compositioncomprising the binding agent of claim 33 and a pharmaceuticallyacceptable carrier.
 40. A method of treating cancer comprisingadministering to a subject in need thereof the binding agent of claim33.